Process for producing cytotoxic lymphocyte

ABSTRACT

The present invention relates to a method for preparing a cytotoxic lymphocyte characterized in that the method comprises the step of carrying out at least one of induction, maintenance and expansion of a cytotoxic lymphocyte in the presence of fibronectin, a fragment thereof or a mixture thereof.

This application is a Divisional of co-pending U.S. application Ser. No.10/509,055 filed on Sep. 24, 2004 and for which priority is claimedunder 35 U.S.C. §120. U.S. application Ser. No. 10/509,055 is thenational phase of PCT International Application No. PCT/JP03/03575 filedon Mar. 25, 2003 under 35 U.S.C. §371. U.S. application Ser. No.10/509,055 also claims the benefit of priority of 2002-84414 under 35U.S.C. §119, which was filed in Japan on Mar. 25, 2002. The entirecontents of each of the above-identified applications are herebyincorporated by reference.

TECHNICAL FIELD

The present invention relates to a method for preparing a cytotoxiclymphocyte, which is useful in the medical field.

BACKGROUND ART

A living body is protected from foreign substances mainly by an immuneresponse, and an immune system has been established by various cells andthe soluble factors produced thereby. Among them, leukocytes, especiallylymphocytes, play a key role. The lymphocytes are classified in twomajor types, B lymphocyte (which may be hereinafter referred to as Bcell) and T lymphocyte (which may be hereinafter referred to as T cell),both of which specifically recognize an antigen and act on the antigento protect the living body.

T cell is subclassified to helper T cell having CD(Cluster Designation)4marker (hereinafter referred to as T_(H)), mainly involved in assistingin antibody production and induction of various immune responses, andcytotoxic T cell having CD8 marker (T_(c): cytotoxic T lymphocyte, alsoreferred to as killer T cell, which may be hereinafter referred to asCTL), mainly exhibiting a cytotoxic activity. CTL, which plays the mostimportant role in recognizing, destroying and eliminating tumor cell,virus-infected cell or the like, does not produce an antibodyspecifically reacting with an antigen like in B cell, but directlyrecognizes and acts on antigens (antigenic peptide) from a target cellwhich is associated with major histocompatibility complex [MHC, whichmay be also referred to as human leukocyte antigen (HLA) in human] ClassI molecules existing on the surface of the target cell membrane. At thistime, T cell receptor (hereinafter referred to as TCR) existing on thesurface of the CTL membrane specifically recognizes the above-mentionedantigenic peptides and MHC Class I molecules, and determines whether theantigenic peptide is derived from itself or nonself. Target cell whichhas been determined to be from nonself is then specifically destroyedand eliminated by CTL.

Recent years, a therapy which would cause a heavier physical burden on apatient, such as pharmacotherapy and radiotherapy, has beenreconsidered, and an interest has increased in an immunotherapy with alighter physical burden on a patient. Especially, there has beenremarked an effectiveness of adoptive immunotherapy in which CTL capableof specifically reacting with an antigen of interest is induced in vitrofrom lymphocyte derived from a human having normal immune function, orthe lymphocyte is expanded without induction, and then transferred to apatient. For instance, it has been suggested that in an animal modeladoptive immunotherapy is an effective therapy for virus infection andtumor [authored by Greenberg, P. D., Advances in Immunology, publishedin 1992; Reusser P., et al., Blood, 78(5), 1373-1380 (1991)]. In thistherapy, it is important to maintain or increase the cell number in astate in which the antigen-specific cytotoxic activity of the CTL ismaintained or enhanced.

In the adoptive immunotherapy as described above, it is necessary toadminister cytotoxic lymphocytes in the number of cells of a givenamount or higher in order to obtain a therapeutic effect. In otherwords, it can be said that it is a major problem to obtain the abovenumber of cells in vitro in a short period of time.

In order to maintain and enhance an antigen-specific cytotoxic activityof CTL, there has been generally employed a method of repeatingstimulation with an antigen of interest when a specific response to anantigen for CTL is induced. However, in this method, the number of CTLfinally obtained may usually be decreased, so that a sufficient numberof cells cannot be obtained.

As a method for preparing T cell which is effective for the treatment ofa disease, there has been known, for instance, adoptive immunotherapyusing tumor-infiltrating lymphocyte (TIL) induced with IL-2 in a highconcentration [N. Engl. J. Med., 316, 1310-1321 (1986); Rosenberg S. A.et al, N. Engl. J. Med., 319(25), 1676-1680 (1988); Ho M. et al., Blood,81(8), 2093-2101 (1993)].

Next, regarding the preparation of the antigen-specific CTL, there hasbeen reported a method for isolating and expanding a CMV-specific CTLclone using self-CMV infected fibroblast and IL-2 [Riddell S. A. et al.,J. Immunol., 146(8), 2795-2804 (1991)] or using anti-CD3 monoclonalantibody (anti-CD3 mAb) and IL-2 [Riddell S. A. et al., J. Immunol.Methods, 128(2), 189-201 (1990)].

Furthermore, WO 96/06929 discloses an REM method (rapid expansionmethod). This REM method is a method for expanding a primary T cellpopulation containing antigen-specific CTL and T_(H) in a short periodof time. In other words, this method is characterized in that a largeamount of T cell can be provided by proliferating individual T cellclones, and that the number of antigen-specific CTL is increased usingan anti-CD3 antibody, IL-2, and PBMC (peripheral blood mononuclear cell)made deficient in an ability for proliferation by irradiation, andEpstein-Barr virus (hereinafter simply referred to as EBV)-infectedcells.

In addition, WO 97/32970 discloses a modified REM method, wherein themethod is a method using as a feeder cell an undifferentiated mammalcell strain expressing a T-cell stimulating component which isdistinguishable from PBMC to reduce an amount of PBMC used.

The lymphokine-activated killer cell (LAK cell) is a functional cellpopulation having an cytotoxic activity, which is obtained by addingIL-2 to peripheral blood (peripheral blood leukocyte), umbilical cordblood, tissue fluid or the like containing lymphocytes, and culturingthe cells in vitro for several days. During the culture, proliferationof the LAK cell is further accelerated by adding an anti-CD3 antibodythereto and culturing the cell. The LAK cell thus obtained has acytotoxic activity non-specifically to various cancer cells and othertargets. The LAK cell is also used in the adoptive immunotherapy in thesame manner as the above-mentioned CTL.

As described above, utilization of IL-2 is essential in the step ofobtaining a cytotoxic lymphocyte, for instance, CTL, LAK cell, TIL orthe like. The cell is further activated by binding IL-2 to interleukin-2receptor (IL-2R) on a cell surface. In addition, IL-2R has been known asan activation marker for a lymphocyte. From these viewpoints, it isimportant to improve IL-2R expression on the cell surface. In addition,in the induction of CTL, it is important to improve an efficiency forinducing a precursor cell of CTL subjected to stimulation by an antigenas CTL, i.e., to improve a proportion (ratio) of the CD8-positive cellin a group of cells after the induction.

Fibronectin is a gigantic glycoprotein having a molecular weight of 250thousands, which exists in an animal blood, on the surface of a culturedcell, or in an extracellular matrix of a tissue, and has been known tohave various functions. A domain structure thereof is divided into sevenportions (hereinafter refer to FIG. 1), wherein three kinds of similarsequences are contained in an amino acid sequence thereof; repetitionsof each of these sequences constituting the entire sequence. Three kindsof the similar sequences are referred to as type I, type II and typeIII. Among them, the type III is constituted by 71 to 96 amino acidresidues, wherein a coincidence ratio of these amino acid residues is 17to 40%. In fibronectin, there are fourteen type III sequences, amongwhich the 8th, 9th or 10th sequence (each being hereinafter referred toas III-8, III-9 or III-10) is contained in a cell binding domain, andthe 12th, 13th or 14th sequence (each being hereinafter referred to asIII-12, III-13 or III-14) is contained in a heparin binding domain. Inaddition, a VLA (very late activation antigen)-5 binding region iscontained in III-10, and its core sequence is RGDS. In addition, aregion referred to as IIICS exists at a C-terminal side of the heparinbinding domain. A region referred to as CS-1 consisting of 25 aminoacids and having a binding activity to VLA-4 exists in IIICS (Deane F.Momer, FIBRONECTIN, ACADEMIC PRESS INC., 1-8 (1988); Kimizuka F. et al.,J. Biochem. 110, 284-291 (1991); Hanenberg H. et al., Human Gene Therapy8, 2193-2206 (1997)).

DISCLOSURE OF INVENTION

An object of the present invention is to a method for preparing acytotoxic lymphocyte having a cytotoxic activity at a high level, whichis suitably used in the medical field.

Concretely, the present invention relates to:

[1] a method for preparing a cytotoxic lymphocyte characterized in thatthe method comprises the step of carrying out at least one of induction,maintenance and expansion of a cytotoxic lymphocyte in the presence offibronectin, a fragment thereof or a mixture thereof;[2] the method according to the above [1], wherein the cytotoxiclymphocyte highly expresses an interleukin-2 receptor as compared to acytotoxic lymphocyte obtained by carrying out at least one of induction,maintenance and expansion in the absence of fibronectin, a fragmentthereof or a mixture thereof;[3] the method according to the above [1], wherein the cytotoxiclymphocyte contains CD8-positive cell in a higher ratio as compared to acytotoxic lymphocyte obtained by carrying out at least one of induction,maintenance and expansion in the absence of fibronectin, a fragmentthereof or a mixture thereof;[4] the method according to any one of the above [1] to [3], wherein thecytotoxic lymphocyte highly maintains cytotoxic activity as compared toa cytotoxic lymphocyte obtained by carrying out at least one ofinduction, maintenance and expansion in the absence of fibronectin, afragment thereof or a mixture thereof;[5] the method according to any one of the above [1] to [4], whereinfibronectin, a fragment thereof or a mixture thereof is immobilized in asolid phase;[6] the method according to the above [5], wherein the solid phase is acell culture equipment or a cell culture carrier;[7] the method according to the above [6], wherein the cell cultureequipment is a petri dish, a flask or a bag, and the cell culturecarrier is beads, a membrane or a slide glass;[8] the method according to any one of the above [1] to [4], wherein atleast one of induction, maintenance and expansion of a cytotoxiclymphocyte is carried out in a medium containing fibronectin, a fragmentthereof or a mixture thereof;[9] the method according to any one of the above [1] to [8], wherein thefibronectin fragment is a polypeptide comprising at least one of theamino acid sequences represented by SEQ ID NOs: 1 to 7 of SequenceListing, or a polypeptide having substitution, deletion, insertion oraddition of one or more amino acids in the amino acid sequence of theabove polypeptide, wherein the polypeptide has functions equivalent tothat of the above polypeptide;[10] the method according to the above [9], wherein the fibronectinfragment has cell adhesion activity and/or heparin binding activity;[11] the method according to the above [9], wherein the fibronectinfragment is a polypeptide selected from polypeptides comprising any oneof the amino acid sequences shown in SEQ ID NOs: 8 to 19 of SequenceListing;[12] the method according to the above [1] comprising carrying out atleast one of induction, maintenance and expansion of a cytotoxiclymphocyte in the presence of fibronectin, a fragment thereof or amixture thereof in a cell culture equipment containing a medium, whereinthe method satisfies any one of the conditions of:

-   (a) a ratio of the number of cells at initiation of culture to a    culture area in the cell culture equipment being 1 to 5×10⁵    cells/cm²; and-   (b) a concentration of cells in a medium at initiation of culture    being 1 to 5×10⁵ cells/ml;    [13] the method according to the above [12], wherein the method    excludes a dilution step or a step of exchanging a cell culture    equipment;    [14] a cytotoxic lymphocyte obtained by the method of any one of the    above [1] to [13];    [15] a medicament comprising as an effective ingredient a cytotoxic    lymphocyte obtained by the method of any one of the above [1] to    [13];    [16] an agent for enhancing an interleukin-2 receptor expression of    a cell, characterized in that the agent comprises as an effective    ingredient fibronectin, a fragment thereof or a mixture thereof;    [17] the agent according to the above [16], wherein the fibronectin    fragment is a polypeptide comprising at least one of the amino acid    sequences represented by SEQ ID NOs: 1 to 7 of Sequence Listing, or    a polypeptide having substitution, deletion, insertion or addition    of one or more amino acids in the amino acid sequence of the above    polypeptide, wherein the polypeptide has functions equivalent to    that of the above polypeptide;    [18] the agent according to the above [17], wherein the fibronectin    fragment has cell adhesion activity and/or heparin binding activity;    [19] the agent according to the above [17], wherein the fibronectin    fragment is a polypeptide selected from polypeptides comprising any    one of the amino acid sequences shown in SEQ ID NOs: 8 to 19 of    Sequence Listing;    [20] an agent for improving a ratio of CD8-positive cell in a    lymphocyte, characterized in that the agent comprises as an    effective ingredient fibronectin, a fragment thereof or a mixture    thereof;    [21] the agent according to the above [20], wherein the fibronectin    fragment is a polypeptide comprising at least one of the amino acid    sequences represented by SEQ ID NOs: 1 to 7 of Sequence Listing, or    a polypeptide having substitution, deletion, insertion or addition    of one or more amino acids in the amino acid sequence of the above    polypeptide, wherein the polypeptide has functions equivalent to    that of the above polypeptide;    [22] the agent according to the above [21], wherein the fibronectin    fragment has cell adhesion activity and/or heparin binding activity;    [23] the agent according to the above [21], wherein the fibronectin    fragment is a polypeptide selected from polypeptides comprising any    one of the amino acid sequences shown in SEQ ID NOs: 8 to 19 of    Sequence Listing;    [24] an agent for improving or maintaining cytotoxic activity in a    cytotoxic lymphocyte, characterized in that the agent comprises as    an effective ingredient fibronectin, a fragment thereof or a mixture    thereof;    [25] the agent according to the above [24], wherein the fibronectin    fragment is a polypeptide comprising at least one of the amino acid    sequences represented by SEQ ID NOs: 1 to 7 of Sequence Listing, or    a polypeptide having substitution, deletion, insertion or addition    of one or more amino acids in the amino acid sequence of the above    polypeptide, wherein the polypeptide has functions equivalent to    that of the above polypeptide;    [26] the agent according to the above [25], wherein the fibronectin    fragment has cell adhesion activity and/or heparin binding activity;    [27] the agent according to the above [25], wherein the fibronectin    fragment is a polypeptide selected from polypeptides comprising any    one of the amino acid sequences shown in SEQ ID NOs: 8 to 19 of    Sequence Listing;    [28] a method for increasing expression of an interleukin-2 receptor    in a cytotoxic lymphocyte, characterized in that the method    comprises the step of carrying out at least one of induction,    maintenance and expansion of a cytotoxic lymphocyte in the presence    of fibronectin, a fragment thereof or a mixture thereof;    [29] a method for improving a ratio of CD8-positive cell in a    cytotoxic lymphocyte, characterized in that the method comprises the    step of carrying out at least one of induction, maintenance and    expansion of a cytotoxic lymphocyte in the presence of fibronectin,    a fragment thereof or a mixture thereof;    [30] a method for improving or maintaining cytotoxic activity in a    cytotoxic lymphocyte, characterized in that the method comprises the    step of carrying out at least one of induction, maintenance and    expansion of a cytotoxic lymphocyte in the presence of fibronectin,    a fragment thereof or a mixture thereof;    [31] the method according to any one of the above [1] to [13],    further comprising the step of transducing a foreign gene into a    cytotoxic lymphocyte; and    [32] the method according to the above [31], wherein the foreign    gene is transduced using retrovirus, adenovirus, adeno-associated    virus or simian virus.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view showing a domain structure of fibronectin.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention has been completed by the findings that in thecytotoxic lymphocyte prepared in the presence of fibronectin and/or afragment thereof, a high cytotoxic activity is maintained, an expressionlevel of IL-2R is significantly increased, and a ratio of theCD8-positive cell is improved.

Incidentally, the preparation of a cytotoxic lymphocyte as used hereinrefers to a step encompassing a step comprising each of the steps ofinduction (activation), maintenance and expansion of the cell, or thecombined steps thereof. The preparation of a cytotoxic lymphocyte of thepresent invention is also referred to culture of a cytotoxic lymphocyte.

The present invention will be explained concretely hereinbelow.

(1) Fibronectin and Fragment Thereof Used in the Present Invention

The fibronectin and a fragment thereof as mentioned herein may be thoseobtained from nature, or those which are artificially synthesized. Thefibronectin and a fragment thereof can be prepared in a substantiallypure form from a substance of natural origin, on the basis of thedisclosure of Ruoslahti E. et al. [J. Biol. Chem., 256(14), 7277-7281(1981)]. The term “substantially pure fibronectin or fibronectinfragment” as referred to herein means that these fibronectin andfibronectin fragment do not substantially contain other proteins and thelike existing together with fibronectin in nature. Each of theabove-mentioned fibronectin and a fragment thereof can be used in thepresent invention alone or in admixture of plural kinds.

The useful information relating to the fibronectin fragments which canbe used in the present invention and the preparation of the fragmentscan be obtained from Kimiduka F., et al. [J. Biochem., 110, 284-291(1991)], Kornbrihtt A. R. et al. [EMBO J., 4(7), 1755-1759 (1985)],Sekiguchi K., et al. [Biochemistry, 25(17), 4936-4941 (1986)], and thelike.

In the present invention, the fibronectin fragment is exemplified by,for instance, a polypeptide comprising an amino acid sequence comprisingat least any of the regions of III-8 (amino acid sequence shown in SEQID NO: 1 in Sequence Listing), III-9 (amino acid sequence shown in SEQID NO: 2 in Sequence Listing), III-10 (amino acid sequence shown in SEQID NO: 3 in Sequence Listing), III-12 (amino acid sequence shown in SEQID NO: 4 in Sequence Listing), III-13 (amino acid sequence shown in SEQID NO: 5 in Sequence Listing), III-14 (amino acid sequence shown in SEQID NO: 6 in Sequence Listing), and CS-1 (amino acid sequence shown inSEQ ID NO: 7 in Sequence Listing) (see FIG. 1).

In addition, as the fragment, a fragment having a cell adhesion activityand/or a heparin binding activity can be preferably used. The celladhesion activity can be evaluated by assaying binding of the fragment(its cell binding domain) used in the present invention to a cell usinga known method. For instance, the method as mentioned above includes amethod of Williams D. A., et al. [Nature, 352, 438-441 (1991)]. Themethod is a method of determining the binding of a cell to a fragmentimmobilized to a culture plate. In addition, the heparin bindingactivity can be evaluated by assaying binding of the fragment (itsheparin binding domain) used in the present invention to heparin using aknown method. For instance, the binding of the fragment to heparin canbe evaluated in the same manner by using heparin, for instance, alabeled heparin in place of the cell in the above-mentioned method ofWilliams D. A. et al.

Further, the fibronectin fragment is exemplified by a polypeptideselected from C-274 (amino acid sequence shown in SEQ ID NO: 8 inSequence Listing), H-271 (amino acid sequence shown in SEQ ID NO: 9 inSequence Listing)., H-296 (amino acid sequence shown in SEQ ID NO: 10 inSequence Listing), CH-271 (amino acid sequence shown in SEQ ID NO: 11 inSequence Listing), CH-296 (amino acid sequence shown in SEQ ID NO: 12 inSequence Listing) or C-CS1 (amino acid sequence shown in SEQ ID NO: 13in Sequence Listing).

Each of the above-mentioned fragments CH-271, CH-296, C-274 and C-CS 1is a polypeptide having a cell binding domain with a binding activity toVLA-5. Also, C-CS1, H-296 or CH-296 is a polypeptide having a cellbinding domain with a binding activity to VLA-4. Further, H-271, H-296,CH-271 or CH-296 is a polypeptide having a heparin binding domain.

In the present invention, a fragment in which each of the above domainsis modified can also be used. The heparin binding domain of thefibronectin is constituted by three type III sequences (III-12, III-13and III-14). A fragment containing a heparin binding domain havingdeletion of one or two of the type III sequences can also be used in thepresent invention. For instance, the fragments may be exemplified byCHV-89 (amino acid sequence shown in SEQ ID NO: 14 of Sequence Listing),CHV-90 (amino acid sequence shown in SEQ ID NO: 15 of Sequence Listing)or CHV-92 (amino acid sequence shown in SEQ ID NO: 16 of SequenceListing), which is a fragment in which a cell binding site of thefibronectin (VLA-5 binding domain: Pro1239 to Ser1515) and one of theIII type sequences are bound, or CHV-179 (amino acid sequence shown inSEQ ID NO: 17 of Sequence Listing) or CHV-181 (amino acid sequence shownin EQ ID NO: 18 of Sequence Listing), which is a fragment in which thecell binding site of the fibronectin and two of the type III sequencesare bound. CHV-89, CHV-90 and CHV-92 contain III-13, III-14 and III-12,respectively, and CHV-179 contains III-13 and III-14, and CHV-181contains III-12 and III-13, respectively.

In addition, a fragment having addition of an additional amino acid toeach of the above-mentioned fragments can be used in the presentinvention. For instance, the fragment can be prepared by adding adesired amino acid to each of the above-mentioned fragment in accordancewith the method for preparing H-275-Cys described in PreparationExamples set forth below. For instance, H-275-Cys (amino acid sequenceshown in SEQ ID NO: 19 of Sequence Listing) is a fragment having aheparin binding domain of the fibronectin, and cysteine residue at aC-terminal.

The fragment used in the present invention may be those comprising apolypeptide comprising an amino acid sequence having substitution,deletion, insertion or addition of one or more amino acids in an aminoacid sequence of a polypeptide constituting a fragment at leastpartially containing an amino acid sequence of naturally occurringfibronectin exemplified above, wherein the polypeptide has an equivalentfunction to the fragment, so long as the desired effects of the presentinvention are obtained.

It is preferable that the substitution or the like of the amino acids iscarried out to an extent that it can change physicochemicalcharacteristics and the like of an inherent polypeptide within the rangethat the function of the polypeptide can be maintained. For instance,the substitution or the like of amino acids is conservative, within therange that the characteristics inherently owned by the polypeptide (forinstance, hydrophobicity, hydrophilicity, electric charge, pK and thelike) are not substantially changed. For instance, the substitution ofthe amino acids is substitutions within each of the groups of {circlearound (1)} glycine, alanine; {circle around (2)} valine, isoleucine,leucine; {circle around (3)} aspartic acid, glutamic acid, asparagine,glutamine; {circle around (4)} serine, threonine; {circle around (5)}lysine, arginine; {circle around (6)} phenylalanine, tyrosine. Deletion,addition or insertion of amino acids is deletion, addition or insertionin the amino acids having characteristics similar to the characteristicsof the surroundings of the subject site in the polypeptide within therange that the characteristics of the surroundings of the subject siteare not substantially changed.

In addition, the phrase “having an equivalent function” refers to thathaving at least any of the functions of (i) a function of maintaining acytotoxic activity of a cytotoxic lymphocyte, (ii) a function ofenhancing an expression level of IL-2R, or (iii) a function of improvinga ratio of CD8-positive cell. Whether or not the fragment comprising apolypeptide having substitution or the like of amino acids has thosefunctions can be appropriately confirmed in accordance with the methoddescribed in Examples set forth below. In addition, as the fragmentcomprising a polypeptide having substitution or the like of amino acids,the fragment having a cell adhesion activity and/or a heparin bindingactivity is preferred. The cell adhesion activity and the heparinbinding activity can be evaluated in accordance with the above-mentionedmethods for determining those activities.

As the fragment comprising a polypeptide having substitution or the likeof amino acids, for instance, a fragment having one or more amino acidsinserted as a linker between two different domains can also be used inthe present invention.

Incidentally, as the fibronectin per se, similarly, there can be used inthe present invention a polypeptide having an amino acid sequence havingsubstitution, deletion, insertion or addition of one or more amino acidsin an amino acid sequence constituting the polypeptide of thefibronectin, wherein the polypeptide has at least any of the functionsof the above-mentioned (i) to (iii).

The fibronectin fragment as referred to herein can also be prepared froma genetic recombinant on the basis of the description of, for instance,U.S. Pat. No. 5,198,423. For instance, each of the fragments of H-271(SEQ ID NO: 9), H-296 (SEQ ID NO: 10), CH-271 (SEQ ID NO: 11) and CH-296(SEQ ID NO: 12) and a method of preparing these fragments are describedin detail in the specification of this patent. In addition, theabove-mentioned C-274 (SEQ ID NO: 8) fragment can be obtained inaccordance with the method described in U.S. Pat. No. 5,102,988.Further, a C-CS1 (SEQ ID NO: 13) fragment can be obtained in accordancewith the method described in Japanese Patent Gazette No. 3104178. Eachof the fragment of CHV-89 (SEQ ID NO: 14), CHV-90 (SEQ ID NO: 15) orCHV-179 (SEQ ID NO: 17) can be obtained in accordance with the methoddescribed in Japanese Patent Gazette No. 2729712. In addition, theCHV-181 (SEQ ID NO: 18) fragment can be obtained in accordance with themethod described in WO 97/18318. The CHV-92 (SEQ ID NO: 16) fragment canbe obtained by genetic engineering technique using a plasmid constructedin a usual manner on the basis of the plasmid described in theseliteratures by referring to Japanese Patent Gazette No. 2729712 and WO97/18318.

These fragments or fragments which can be derived from these fragmentsin a usual manner can be prepared by using microorganisms deposited tothe International Patent Organism Depositary, National Institute ofAdvanced Industrial Science and Technology, Tsukuba Central 6, 1-1,Higashi 1-chome, Tsukuba-shi, Ibaraki-ken, Japan (Zip code 305-8566)under the following accession numbers, or by modifying a plasmid carriedin each microorganism in accordance with a known method.

-   FERM BP-2264 (Escherichia coli carrying a plasmid encoding H-271,    Date of Deposit: Jan. 30, 1989);-   FERM BP-2800 (Escherichia coli carrying a plasmid encoding CH-296,    Date of Deposit: May 12, 1989);-   FERM BP-2799 (Escherichia coli carrying a plasmid encoding H-271,    Date of Deposit: May 12, 1989);-   FERM BP-7420 (Escherichia coli carrying a plasmid encoding H-296,    Date of Deposit: May 12, 1989);-   FERM BP-1915 (Escherichia coli carrying a plasmid encoding C-274,    Date of Deposit: Jun. 17, 1988);-   FERM BP-5723 (Escherichia coli carrying a plasmid encoding C-CS1,    Date of Deposit: Mar. 5, 1990);-   FERM P-12182 (Escherichia coli carrying a plasmid encoding CHV-89,    Date of Deposit: Apr. 8, 1991); and-   FERM P-12183 (Escherichia coli carrying a plasmid encoding CHV-179,    Date of Deposit: Apr. 8, 1991).

Since the fibronectin is a gigantic glycoprotein, it is not necessarilyeasy to prepare and use a naturally occurring protein for the industrialpurpose and for the purpose of the preparation of the medicament.Further, the fibronectin exists in a large amount in plasma in a livingbody. Therefore, when a fibronectin obtained from plasma is used as ablood preparation, there is a risk of contamination of components otherthan the fibronectin, so that there is considered to have a problem fromthe aspect of safety. In addition, since the fibronectin is amultifunctional protein, there may be considered some disadvantagescaused by a region different from the region exhibiting the effect bythe method of the present invention depending on the circumstances ofits use. For these reasons, a fibronectin fragment can be preferablyused in the present invention, more preferably a recombinant fibronectinfragment obtained as described above can be used from the viewpoints ofavailability, easy handling and safety. Further, there can be especiallypreferably used a fibronectin fragment which can exhibit an effect suchas improvement in an expansion ratio of a lymphocyte, increase in anexpression level of IL-2R in an expanded lymphocyte, or improvement in aratio of CD8-positive cell in an expanded lymphocyte population asdescribed below. In addition, the molecular weight of the fibronectinfragment used in the present invention is, but not particularly limitedto, preferably from 1 to 200 kD, more preferably from 5 to 190 kD, evenmore preferably from 10 to 180 kD.

(2) Method for Preparing Cytotoxic Lymphocyte of the Present Invention

The method for preparing the cytotoxic lymphocyte of the presentinvention will be concretely explained below. The method of the presentinvention is a method for preparing cytotoxic lymphocyte comprising thestep of carrying out at least any one of induction, maintenance andexpansion of a cytotoxic lymphocyte in the presence of theabove-mentioned fibronectin, a fragment thereof or a mixture thereof.

The “cytotoxic lymphocyte” as used herein means a group of cellscontaining a cytotoxic lymphocyte. In a narrow sense, the cytotoxiclymphocyte may be referred only to a cytotoxic lymphocyte contained inthe above-mentioned group of cells in some cases. In addition, thepreparation of the cytotoxic lymphocyte in the present inventionencompasses any of induction from a precursor cell which can be formedinto the lymphocyte of the present invention to a lymphocyte having acytotoxic activity, maintenance of the cytotoxic lymphocyte, andexpansion of the cytotoxic lymphocyte using the cytotoxic lymphocyteand/or the precursor cell.

The cytotoxic lymphocyte of the present invention includes, but notparticularly limited to, for instance, cytotoxic T cell (CTL),lymphokine-activated killer cell (LAK cell), tumor-infiltratinglymphocyte (TIL), NK cell and the like, each having an antigen-specificcytotoxic activity.

In the present invention, the precursor cell which can be formed into acytotoxic lymphocyte, i.e., the precursor cell which has an ability ofdifferentiating into the lymphocyte, is exemplified by PBMC, NK cell,naive cell, memory cell, hemopoietic stem cell, umbilical cord bloodmononuclear cell and the like. In addition, so long as a cell is ahemocyte, the cell can be used as a precursor cell in the presentinvention. Any of these cells which are collected from a living body canbe used directly or those which are subjected to frozen storage can beused. Incidentally, in the method for preparing a cytotoxic lymphocyteof the present invention, a material containing the above-mentionedcells, for instance, a blood such as peripheral blood or umbilical cordblood; one obtained by removing components such as erythrocyte andplasma from the blood; a marrow fluid and the like can be used.

One of the major characteristics of the method for preparing a cytotoxiclymphocyte of the present invention resides in that the cytotoxiclymphocyte is prepared in the presence of an effective ingredientselected from fibronectin, a fragment thereof or a mixture thereof.

In the method of the present invention, the induction, maintenanceand/or expansion of the cytotoxic lymphocyte is usually performed in amedium containing given components in the presence of theabove-mentioned effective ingredient of the present invention.

For instance, in the method of the present invention, when the inductionor expansion of the cytotoxic lymphocyte is intended, the number ofcells (cytotoxic lymphocytes and/or precursor cells) at the initiationof culture used in the present invention is not particularly limited.For instance, the number is preferably from 1 to 1×10⁸ cells/ml. Inaddition, the culture conditions are not particularly limited, and usualconditions for cell culture can be employed. For instance, cells can becultured under the conditions of 37° C. in the presence of 5% CO₂ andthe like. In addition, the medium can be exchanged with a fresh mediumat appropriate intervals.

The medium used in the method for preparing a cytotoxic lymphocyte ofthe present invention is not particularly limited, and a known mediumprepared by mixing components necessary for maintaining and growing acytotoxic lymphocyte or its precursor cell can be used. For instance, acommercially available medium may be used. These media may containappropriate proteins, cytokines and other components in addition to theinherent constituents. Preferably, a medium containing IL-2 is used inthe present invention. The concentration of IL-2 in the medium is, butnot particularly limited to, for instance, preferably from 0.01 to 1×10⁵U/ml, more preferably from 0.1 to 1×10⁴ U/ml.

In addition, a precursor cell which can be formed into a cytotoxiclymphocyte can be co-cultured in a medium further containing an anti-CD3antibody. The concentration of the anti-CD3 antibody in a medium is, butnot particularly limited to, for instance, preferably from 0.01 to 100μg/ml. The anti-CD3 antibody can be added for the purpose of activatinga receptor on a lymphocyte. Also, besides the above, alymphocyte-stimulating factor such as lecithin can be added. Theconcentration of the component in a medium is not particularly limited,so long as the desired effects can be obtained.

Besides the coexistence of these components by dissolving the componentsin a medium, there may be used by immobilization to an appropriate solidphase, for instance, a cell culture equipment (including any of those ofopen system and closed system), such as a petri dish, a flask or a bag,or to a cell culture carrier such as beads, a membrane or a slide glass.The materials for those solid phases are not particularly limited solong as the materials can be used for cell culture. When the componentsare immobilized to, for instance, the above-mentioned equipment, it ispreferable to immobilize a given amount of each component to the amountof the medium to be placed in the equipment so that the medium has asimilar proportion to a desired concentration of the case where thecomponents are used by dissolving the components in a medium uponplacing the medium in the equipment. The amount of the componentsimmobilized is not particularly limited, so long as the desired effectscan be obtained. The above-mentioned carrier is used by immersing thecarrier in a culture medium in the cell culture equipment during thecell culture. When the above-mentioned components are immobilized to theabove-mentioned carrier, it is preferable to immobilize a given amountof each component to the amount of the medium to be placed in theequipment so that the medium has a similar proportion to a desiredconcentration of the case where the components are used by dissolvingthe components in a medium upon placing the carrier in the medium. Theamount of the components immobilized is not particularly limited, solong as the desired effects can be obtained.

In both cases, the immobilization of the above-mentioned components canbe carried out by a known method, for instance, a method forimmobilizing a fibronectin fragment set forth below.

Furthermore, there may be used together with the above-mentionedcomponents a compound selected from the group consisting of acidicpolysaccharides, acidic oligosaccharides, acidic monosaccharides andsalts thereof which are effective for induction of a cytotoxic T cellhaving an antigen-specific cytotoxic activity, described in WO 02/14481,or a substance selected from the following (A) to (D):

-   (A) a substance having a binding activity to CD44;-   (B) a substance capable of regulating a signal emitted by binding a    CD44 ligand to CD44;-   (C) a substance capable of inhibiting binding of a growth factor to    a growth factor receptor; and-   (D) a substance capable of regulating a signal emitted by binding of    a growth factor to a growth factor receptor.

The above-mentioned substance having a binding activity to CD44 isexemplified by, for instance, a CD44 ligand and/or an anti-CD44antibody. The substance capable of regulating a signal emitted bybinding a CD44 ligand to CD44 includes, for instance, various inhibitorsfor phosphoenzymes. The substance capable of inhibiting binding of agrowth factor to a growth factor receptor includes, for instance, asubstance having a binding activity to a growth factor and forming acomplex with the growth factor, thereby inhibiting the binding of thegrowth factor to a growth factor receptor, or a substance having abinding activity to a growth factor receptor, thereby inhibiting thebinding of the growth factor to a growth factor receptor. Furthermore,the substance capable of regulating a signal emitted by binding of agrowth factor to a growth factor receptor includes, for instance,various inhibitors for phosphoenzymes. The concentration of thesecomponents in the medium is not particularly limited, so long as thedesired effects can be obtained. Also, these components may be used byimmobilization to the appropriate solid phase as mentioned above inaddition to the coexistence of these components in the medium bydissolving the components in the medium.

Here, each of various substances mentioned above can be used alone or inadmixture of two or more kinds.

In the present invention, the phrase “in the presence of theabove-mentioned effective ingredient” refers to the fact that theeffective ingredient is present in a state that the above-mentionedeffective ingredient can exhibit its function when the induction,maintenance and expansion of the cytotoxic lymphocyte is carried out,and the existing manner is not particularly limited. For instance, whenthe effective ingredient is dissolved in the medium to be used, thecontent of the effective ingredient of the present invention in themedium in which co-culture is carried out is not particularly limited,so long as the desired effects are obtained. The content of theeffective ingredient is, for instance, preferably from 0.01 to 1000μg/ml, more preferably from 0.1 to 1000 μg/ml, even more preferably from1 to 100 μg/ml. Besides the coexistence of the effective ingredient bydissolving the effective ingredient in a medium as above, there may beused by immobilization to an appropriate solid phase, for instance, acell culture equipment (including any of those of open system and closedsystem), such as a petri dish, a flask or a bag, or to a cell culturecarrier such as beads, a membrane or a slide glass. From the viewpointof administering the cultured cytotoxic lymphocyte to a living body, itis desired that the above-mentioned effective ingredient is immobilized,but it is not particularly limited thereto.

Once various components mentioned above or the effective ingredient ofthe present invention is immobilized to the solid phase, the cytotoxiclymphocyte can be easily separated from the effective ingredient or thelike after the lymphocyte is obtained by the method of the presentinvention only by separating the lymphocyte from the solid phase, sothat the contamination of the effective ingredient into the lymphocytecan be prevented.

When the effective ingredient of the present invention is immobilizedto, for instance, the above-mentioned equipment, it is preferable toimmobilize a given amount of each effective ingredient to the amount ofthe medium to be placed in the equipment so that the medium has asimilar proportion to a desired concentration in a case where theeffective ingredient is used by dissolving the effective ingredient in amedium upon placing the medium in the equipment. The amount of theeffective ingredient is not particularly limited, so long as the desiredeffects are obtained. When the effective ingredient is immobilized tothe above-mentioned carrier, it is preferable to immobilize a givenamount of each effective ingredient to the amount of the medium to beplaced into a equipment so that the medium has a similar proportion to adesired concentration in a case where the effective ingredient is usedby dissolving the effective ingredient in a medium upon placing thecarrier into the medium. The amount of the effective ingredient is notparticularly limited, so long as the desired effects are obtained.

For instance, the immobilization of the fibronectin fragment can becarried out in accordance with the methods described in WO 97/18318 andWO 00/09168.

When the expression level of IL-2R is determined for the cytotoxiclymphocyte obtained by the method of the present invention, asignificant increase in expression level of IL-2R is recognized ascompared to a cytotoxic lymphocyte obtained by carrying out at least anyone of induction, maintenance and expansion in the absence offibronectin, a fragment thereof or a mixture thereof. Here, theexpression level of IL-2R can be determined by a known method, forinstance, using an anti-IL-2R antibody.

As described above, the cytotoxic lymphocyte obtained by the method ofthe present invention has an increased expression level of IL-2R. IL-2Ris an activation marker which is expressed on a surface of an activatedT cell, and with the expression of this molecule, cytokine production,cytotoxic activity, proliferation activation or the like is activated.Therefore, the cytotoxic lymphocyte obtained by the method of thepresent invention is a group of cells having a high function.

In addition, since the cytotoxic lymphocyte obtained by the method ofthe present invention has an increased expression level of IL-2R, thecytotoxic lymphocyte has an increased sensitivity to a stimulation byIL-2 added to a medium, or IL-2 produced by a precursor cell of acytotoxic lymphocyte, a lymphocyte itself or other coexisting cell. Forthis reason, the cytotoxic lymphocyte can be activated by itself evenunder the environment of a smaller amount of IL-2 (for instance, in aliving body or the like).

Further, in the cytotoxic lymphocyte obtained by the method of thepresent invention, the existence ratio of (CD8-positive) cell having aCD8 marker is high as compared to that of the cytotoxic lymphocyteobtained by carrying out at least any one of induction, maintenance andexpansion in the absence of fibronectin, a fragment thereof or a mixturethereof. This fact has some advantages, for instance, {circle around(1)} that the CD8-positive cell produces a cytokine such asinterferon-γ, thereby causing immunological activation to change ahelper T cell balance into the Th1 dominant system, {circle around (2)}that the CD8-positive cell is a cellular immunocyte that can efficientlyexclude a foreign substance such as a virus or a tumor cell, {circlearound (3)} that when the CD8-positive cell is obtained, the

CD8-positive cell can be enriched with culturing the cell in accordancewith the method of the present invention, while the CD8-positive cellhas been conventionally purified with magnet beads or a flow cytometer,{circle around (4)} that the cytotoxic lymphocyte is suitably used as aprecursor cell during the induction of CTL, because the ratio of theCD8-positive cell is high, {circle around (5)} that even a cellpopulation having a lower ratio of the CD8-positive cell can be culturedwith increasing the ratio of the CD8-positive cell and the like.Therefore, the method of the present invention is very useful in thepreparation of a cytotoxic lymphocyte.

Here, the ratio of the CD8-positive cell in the cytotoxic lymphocyteobtained by the method of the present invention can be determined by,for instance, but not particularly limited to, using an anti-CD8antibody.

In addition, the cytotoxic lymphocyte, especially CTL, preparedaccording to the method of the present invention has an excellentcharacteristic that there is no drastic decrease in cytotoxic activityas previously observed, even when a cell after the culture is maintainedover a long period of time, or the cell is proliferated. In other words,the cytotoxic lymphocyte maintains a high cytotoxic activity as comparedto a cytotoxic lymphocyte obtained by carrying out at least any one ofinduction, maintenance and expansion in the absence of fibronectin, afragment thereof or a mixture thereof. Therefore, there can bemaintained as a lymphocyte having a stable cytotoxic activity by cloningthe cultured cytotoxic lymphocyte. In addition, the induced CTL can beproliferated and expanded by stimulating the CTL with an antigen,various kinds of cytokines, or an anti-CD3 antibody. A known method canbe used for the maintenance or expansion of the cytotoxic lymphocytewithout being particularly limited.

The maintenance of the above-mentioned cytotoxic lymphocyte refers tothe maintenance of the cytotoxic lymphocyte with keeping its cytotoxicactivity. The culture conditions during the maintenance are notparticularly limited, and the conditions used for ordinary cell culturecan be used. For instance, the cells can be cultured under theconditions of 37° C. in the presence of 5% CO₂, and the like. Inaddition, the medium can be exchanged with a fresh one at appropriatetime intervals. The medium to be used and other componentssimultaneously used therewith and the like are the same as thosementioned above.

One of the major characteristics of the maintenance and expansion of thecytotoxic lymphocyte in the method of the present invention resides inthat the method comprises respectively continuously culturing andexpanding the cytotoxic lymphocyte in a medium in the presence of theeffective ingredient of the present invention, i.e. fibronectin, afragment thereof or a mixture thereof. According to the expansion, thecell number of the cytotoxic lymphocyte can be increased in a state thatthe cytotoxic activity owned by the cytotoxic lymphocyte is maintained.In other words, as one embodiment of the method of the presentinvention, there is provided a method for expanding a cytotoxiclymphocyte.

In the method for expanding a cytotoxic lymphocyte of the presentinvention, the culture conditions are not particularly limited, and theconditions used for ordinary cell culture can be used. For instance, thecells can be cultured under the conditions at 37° C. in the presence of5% CO₂, and the like. In addition, the medium can be exchanged with afresh one at appropriate time intervals. The medium to be used and othercomponents simultaneously used therewith and the like are the same asthose mentioned above.

According to the method for expansion of the present invention, forinstance, in the case of the expansion of CTL, CTL of which cell numberis increased 100- to 1000-folds can be obtained by expansion for 14days. In addition, as one example of the case of expansion of LAK cell,there can be obtained LAK cell increased in about 200-folds by culturingthe cell for 7 days, and in about 1000-folds by culturing the cell for 9days. Further, the thus obtained cytotoxic lymphocyte, especially CTL,has a higher cytotoxic activity as compared to one obtained by aconventional method for expanding a cytotoxic lymphocyte, for instance,a REM method or a modified REM method. The effects of the presentinvention as described above can be confirmed by determining a cytotoxicactivity owned by CTL or the like expanded by the method of the presentinvention, in accordance with the method described in Examples set forthbelow.

Further, the method for preparing a cytotoxic lymphocyte of the presentinvention has the feature that the culture can be initiated at a lownumber of cells. A large amount of lymphocytes is required in order tocarry out adopted immunotherapy, but it is difficult to obtain a largeamount of lymphocytes from a patient. In addition, in an ordinaryexpansion of the cytotoxic lymphocyte, there have been necessitatedselection of a cell culture equipment having an appropriate culture areadepending upon the number of cells to be used, and culture at anappropriate amount of the medium. In other words, usually, the cultureis initiated under the high density conditions that the amount (number)of cells to a culturing area in a cell culture equipment [i.e. area(cm²) of a surface area of the equipment contacting with the medium] is1×10⁶ cells/cm² or more, and the cell concentration is 1×10⁶ cells/ml ormore. When the culture is carried out under the conditions below thiscell level, an expansion ratio [a ratio of the number of cells after theexpansion to the number of cells before the expansion (number of cellsafter expansion/number of cells before expansion)] becomes very low,whereby requiring a long-term culture period before the cytotoxiclymphocytes are obtained in a large amount. Therefore, generally, alarge number of lymphocytes are currently prepared by, for instance,initiating the culture using a small cell culture equipment, andthereafter using a stepwise, large-scaled cell culture equipment, or amethod of increasing the number of cell culture equipments and repeatingdilution procedures. As described above, a plurality of culture systemsare required in the ordinary expansion of the cytotoxic lymphocyte.

According to the method of the present invention, even when initiatedwith a small amount of cells, the cell can be cultured with a highexpansion ratio regardless of the size of a cell culture equipment.Therefore, a complicated procedure such as an exchange of the cellculture equipment and the dilution procedures as described above becomeunnecessary. In other words, according to the method of the presentinvention, the expansion of the cytotoxic lymphocyte can besatisfactorily carried out by culture procedures using one cell cultureequipment, i.e., one culture system. Therefore, the method of thepresent invention is a method for preparing a cytotoxic lymphocyte whichexcludes the dilution step or the step of exchanging a cell cultureequipment. Especially, when LAK cell is expanded according to the methodof the present invention, LAK cell can be expanded by adding a cellwhich can be formed into a LAK cell and a medium to a large-volume cellculture equipment, and adding only IL-2 thereto in subsequent steps. Thepresent invention is very useful in the aspect that a large amount ofLAK cell can be obtained by a simple procedure. Here, the fibronectinfragment can be preferably used as the effective ingredient of thepresent invention to be used from the viewpoint of obtaining a higherexpansion ratio. As described above, according to the method of thepresent invention, a necessary amount of the cytotoxic lymphocyte can beobtained in a shorter time period.

For instance, when at least any one of induction, maintenance andexpansion of a cytotoxic lymphocyte is initiated at a low number ofcells in a cell culture equipment containing a medium in the presence ofthe effective ingredient of the present invention, the induction,maintenance or expansion can be carried out by using an amount of thecell satisfying the conditions selected from the followings (a) and (b)at the initiation of culture:

(a) the ratio of the amount of cells to the culture area in the cellculture equipment to be used being preferably from 1 to 5×10⁵ cells/cm²,more preferably from 10 to 1×10⁵ cells/cm², especially preferably from1×10² to 5×10⁴ cells/cm²; and(b) the concentration of the cells in the medium being preferably from 1to 5×10⁵ cells/ml, more preferably from 10 to 1×10⁵ cells/ml, especiallypreferably from 1×10² to 5×10⁴ cells/ml.

The amount of cells as used herein refers to the number of cytotoxiclymphocytes and/or precursor cells.

In addition, in the method of the present invention, there can beexemplified a method comprising carrying out at least any one ofinduction, maintenance and expansion of a cytotoxic lymphocyte in oneculturing system, which excludes the step of exchange of a cell cultureequipment or the step of the dilution procedure.

The method of the present invention will be explained by taking thepreparation of CTL as an example.

The induction of CTL is carried out by incubating (culturing) aprecursor cell capable of differentiating to CTL together with anappropriate antigen-presenting cell in the presence of theabove-mentioned effective ingredient in, for instance, any of medium, inorder to give CTL an ability of recognizing the desired antigen. Theprecursor cell is not particularly limited, so long as the precursorcell is a cell which is in a stage before the cell becomes CTL and fatedto differentiate to CTL, and includes, for instance, peripheral bloodmononuclear cell (PBMC), naive cell, memory cell, umbilical cord bloodmononuclear cell, hemopoietic stem cell, and the like. Theantigen-presenting cell is not particularly limited, so long as the cellhas an ability to present an antigen to be recognized to T cell. Forinstance, mononuclear cell, B cell, T cell, macrophage, dendritic cell,fibroblast or the like which is allowed to present a desired antigen canbe used in the present invention.

In the present invention, the culture conditions for a precursor cell orthe like during the preparation of CTL may be, for instance, set inaccordance with generally known conditions [see, for instance, Carter J.et al., Immunology 57(1), 123-129 (1986)].

In addition, the cell can be co-cultured with an appropriate feedercell. When the CTL is co-cultured with the feeder cell, it is desiredthat the medium is one that is suitable for maintenance and growth ofboth the CTL and the feeder cell. As the medium, a commerciallyavailable medium can be used.

The feeder cell used for the method of the present invention is notparticularly limited, so long as the feeder cell stimulates CTLcooperatively with an anti-CD3 antibody to activate T cell receptor. Inthe present invention, for instance, PBMC or B cell transformed withEpstein-Barr virus (EBV-B cell) is used. Usually, a feeder cell is usedafter its proliferating ability is taken away by means of irradiation orthe like. Incidentally, the content of the feeder cell in the medium maybe determined according to the known conditions. For instance, thecontent is preferably from 1×10⁵⁻⁷ cells/ml.

In a particularly preferred embodiment of the present invention,non-virus-infected cell, for instance, a cell other than EBV-B cell, isused as a feeder cell. By using the non-virus-infected cell, thepossibility that EBV-B cell is admixed in an expanded CTL can beeliminated, thereby making it possible to increase the safety in medicaltreatments utilizing CTL, such as adoptive immunotherapy.

The antigen-presenting cell can be prepared by adding an antigenicpeptide to a cell having an antigen-presenting ability, thereby allowingthe cell to present the antigenic peptide on its surface [see, forinstance, Bendnarek M. A. et al., J. Immunol. 147(12), 4047-4053(1991)]. In addition, in the case where a cell having anantigen-presenting ability has an ability to process an antigen, anantigen is added to the cell, whereby the antigen is incorporated intothe cell and processed therein, and fragmented antigenic peptides arepresented on the cell surface. Incidentally, when an antigenic peptideis added to a cell having an antigen-presenting ability, an antigenicpeptide matching the MHC restriction of the antigen-presenting cell usedand the CTL to be induced are used.

Incidentally, the antigen used in the present invention is notparticularly limited, and includes, for instance, exogenous antigenssuch as bacteria and viruses, endogenous antigens such astumor-associated antigens (cancer antigens), and the like.

In the present invention, it is preferable that the antigen-presentingcell is made non-proliferative. In order to make the cellnon-proliferative, the cell may be, for instance, subjected toirradiation with X-ray or the like, or a treatment with an agent such asmitomycin.

In the present invention, common conditions for incubating(co-culturing) a precursor cell capable of differentiating to CTLtogether with an antigen-presenting cell in the presence of theeffective ingredient selected from fibronectin, a fragment thereof or amixture thereof to induce CTL may be known conditions [see, forinstance, Bendnarek M. A. et al., J. Immunol., 147(12), 4047-4053(1991)]. The co-culture conditions are not particularly limited, and theconditions usually used for the cell culture can be used. For instance,the cells can be cultured under the conditions of 37° C. in the presenceof 5% CO₂, and the like. The co-culture is usually carried out for about2 to about 15 days, during which time the antigen-presenting cell may beexchanged with freshly prepared one for restimulation. In addition, themedium can be exchanged with a fresh one at appropriate time intervals.

The CTL thus obtained by the method of the present invention has anability of specifically recognizing the desired antigen, for instance,specifically destroying a cell having the antigen by its cytotoxicactivity. This cytotoxic activity of the CTL can be evaluated by a knownmethod. For instance, the cytotoxic activity of the CTL against a targetcell labeled with a radioactive substance, a fluorescent substance orthe like can be evaluated by determining radioactivity or fluorescentintensity ascribed to the target cell destroyed by the CTL. In addition,there can be detected by determining the amount of a cytokine such asGM-CSF or IFN-γ released antigen-specifically from the CTL or a targetcell. Besides them, the cytotoxic activity can be directly confirmed byusing an antigenic peptide-MHC complex in which the peptide is labeledwith a fluorescent pigment or the like. In this case, for instance, theCTL is contacted with a first fluorescent marker coupled with aCTL-specific antibody, and then with an antigenic peptide-MHC complex inwhich the peptide is coupled with a second fluorescent marker, and thepresence of a double-labeled cell is detected by FACS(fluorescence-activated cell sorting) analysis, whereby the cytotoxicactivity of CTL can be evaluated.

Incidentally, the method for expanding CTL of the present invention isnot particularly limited, so long as the above-mentioned effectiveingredient exists in the culture system used in the method. The presentinvention also encompasses an embodiment in which the method is carriedout by the existence of the above-mentioned effective ingredient in theculture system in a conventional method for expansion of CTL other thanthose described above, i.e. the culture of a precursor cell or the likeis carried out in the presence of the effective ingredient of thepresent invention (for instance, by adding the above-mentioned effectiveingredient to a medium to be used in the culture).

Next, the method for culturing LAK cell will be explained in detail.

The culture of LAK cell is carried out by incubating a cell which can beformed into LAK cell together with IL-2 in the presence of theabove-mentioned effective ingredient. The cell which can be formed intoLAK cell includes, but not particularly limited to, for instance,peripheral blood mononuclear cell (PBMC), NK cell, umbilical cord bloodmononuclear cell, hemopoietic stem cell, blood components containingthese cells, and the like.

In addition, the general conditions for culturing LAK cell may be set inaccordance with the known conditions [for instance, see SaiboKogaku(Cell Technology), 14(2), 223-227 (1995); Saibo Baiyo(CellCulture) 17(6), 192-195 (1991); THE LANCET, 356, 802-807 (2000); CurrentProtocols in Immunology, supplement 17, UNIT 7.7]. The co-cultureconditions are not particularly limited, and the conditions which areused in ordinary cell culture can be employed. For instance, the culturecan be carried out under the conditions of 37° C. in the presence of 5%CO₂, and the like This co-culture is usually carried out for about 2 toabout 15 days. In addition, the medium may be exchanged with a fresh oneat appropriate time intervals.

In the same manner as those for the above-mentioned induction,maintenance or expansion of the CTL or the LAK cell, as to TIL, a groupof cells having a high cytotoxic activity can be prepared by culturingthe cells in the presence of fibronectin, a fragment thereof or amixture thereof. In the present invention, there are no particularlimitation in the procedures of activating these cells so long asfibronectin, a fragment thereof or a mixture thereof is coexistenttherewith, and the procedures can be carried out using a mediumappropriate for culture or activation of the above-mentioned cells. Asto the amount of fibronectin, a fragment thereof or a mixture thereofused, the method of adding the component and the like, appropriate onesmay be selected in accordance with the above-mentioned method.

According to the above-mentioned method for preparing a cytotoxiclymphocyte of the present invention, there is obtained a cytotoxiclymphocyte in which a cytotoxic activity is maintained at a high level,an expression level of IL-2R is significantly increased, and a ratio ofa CD8-positive cell is improved, the cytotoxic lymphocyte which issuitable for use in medicine. Accordingly, as one embodiment of themethod of the present invention, there are further provided a method forincreasing expression of an interleukin-2 receptor in a cytotoxiclymphocyte, comprising the step of carrying out at least any one ofinduction, maintenance and expansion of a cytotoxic lymphocyte in thepresence of the effective ingredient of the present invention; a methodfor improving a ratio of CD8-positive cell in a cytotoxic lymphocyte,comprising the step of carrying out any one of induction, maintenanceand expansion of a cytotoxic lymphocyte in the presence of the effectiveingredient of the present invention; and a method for improving ormaintaining a cytotoxic activity in a cytotoxic lymphocyte, comprisingthe step of carrying out at least any one of induction, maintenance andexpansion of a cytotoxic lymphocyte in the presence of the effectiveingredient of the present invention.

In another embodiment of the present invention, there is provided anagent for enhancing IL-2R expression on a cell surface, which comprisesas an effective ingredient fibronectin, a fragment thereof or a mixturethereof. The enhancing agent comprises the effective ingredient itselfand further other optional ingredient, for instance, a medium, aprotein, and a cytokine (preferably IL-2) which are appropriate for acell to be activated, and other desired components. Also, the mediumcontaining the above-mentioned enhancing agent can be employed as amedium for enhancing IL-2R expression in a cytotoxic lymphocyte. Theabove-mentioned medium optionally contains basic components for the cellculture. Here, the enhancing agent and the medium for enhancing IL-2Rexpression mentioned above can be prepared using the effectiveingredient of the present invention in accordance with a known method.The content of the effective ingredient of the present invention and thelike in the enhancing agent or the medium for enhancing IL-2R expressionmentioned above is not particularly limited, so long as the desiredeffects of the present invention are obtained. For instance, the contentcan be appropriately determined in accordance with the content of theeffective ingredient and the like in the above-mentioned medium used inthe method of the present invention as desired. In addition, theabove-mentioned enhancing agent may be directly administered to a livingbody, whereby IL-2R expression on a cell in a living body can beenhanced.

In still another embodiment of the present invention, there is providedan agent for improving a ratio of CD8-positive cell in a culturedlymphocyte population, characterized in that the agent comprises as aneffective ingredient fibronectin, a fragment thereof or a mixturethereof. The ratio-improving agent comprises the effective ingredientitself and further other optional ingredient, for instance, a medium, aprotein, and a cytokine (preferably IL-2) which are appropriate for acell to be activated, and other desired components. Also, the mediumcontaining the above-mentioned ratio-improving agent can be employed asa medium for improving a ratio of CD8-positive cell in a cytotoxiclymphocyte. The above-mentioned medium optionally contains basiccomponents for the cell culture. Here, the ratio-improving agent and themedium for improving the ratio mentioned above can be prepared using theeffective ingredient of the present invention in accordance with a knownmethod. The content of the effective ingredient of the present inventionand the like in the ratio-improving agent or the medium for improving aratio of CD8-positive cell mentioned above can be appropriatelydetermined as desired in the same manner as in the case of theabove-mentioned agent for expressing IL-2R and the like. In addition,the above-mentioned ratio-improving agent may be directly administeredto a living body, whereby the ratio of a cytotoxic lymphocyte in aliving body can be improved.

In another embodiment of the present invention, there is provided anagent for improving or maintaining cytotoxic activity in a cytotoxiclymphocyte, characterized in that the agent comprises as an effectiveingredient fibronectin, a fragment thereof or a mixture thereof. Theimproving or maintaining agent comprises the effective ingredient itselfand further other optional ingredient, for instance, a medium, aprotein, and a cytokine (preferably IL-2) which are suitable for a cellto be activated, and other desired components. Also, the mediumcontaining the above-mentioned improving or maintaining agent can beemployed as a medium for improving or maintaining a cytotoxic activityin a cytotoxic lymphocyte. The above-mentioned medium optionallycontains basic components for the cell culture. Here, the improving ormaintaining agent and the medium for improvement or maintenancementioned above can be prepared using the effective ingredient of thepresent invention in accordance with a known method. The content of theeffective ingredient of the present invention in the improving ormaintaining agent and the medium for improvement or maintenancementioned above are not particularly limited so long as the desiredeffects of the present invention are obtained. For instance, the contentcan be appropriately determined in accordance with the content of theeffective ingredient in the medium mentioned above used in the method ofthe present invention as desired. In addition, the above-mentionedimproving or maintaining agent may be directly administered to a livingbody, whereby the activity of the cytotoxic lymphocyte in a living bodycan be improved or maintained.

Furthermore, the expression-enhancing agent, the ratio-improving agentand the agent for improving or maintaining a cytotoxic activitymentioned above may be in the form in which the components areimmobilized to an appropriate solid phase, for instance, a cell cultureequipment such as a petri dish, a flask or a bag (including both ofthose of an open system and closed system), and a cell culture carriersuch as beads, a membrane or a slide glass.

Usually, in the lymphocyte-containing culture obtained by using themethod for preparing a cytotoxic lymphocyte as described above, cellsother than cytotoxic lymphocyte such as helper T cell are admixedtherein. However, since lymphocytes having a cytotoxic activity arecontained in a large amount in the lymphocyte-containing cultureobtained by the present invention, the cells in the culture can beharvested from the culture by centrifugation or the like, and directlyused as a cytotoxic lymphocyte obtained by the method of the presentinvention. Moreover, if the above-mentioned effective ingredient or thelike is immobilized to a cell culture equipment or the like, there is norisk of admixture of the component or the like in the resultingcytotoxic lymphocyte.

In addition, a cell population (or culture) rich in a cytotoxiclymphocyte can be further separated from the culture by a known method,and used as a cytotoxic lymphocyte of the present invention. In otherwords, the method for preparing a cytotoxic lymphocyte obtained by themethod of the present invention can comprise the step of selecting acell population rich in a cytotoxic lymphocyte from the culture obtainedby the method.

The method of selecting a cell population rich in a cytotoxic lymphocyteis not particularly limited. The method is exemplified by, for instance,a method comprising selectively collecting only the desired cell fromthe culture using a cell culture equipment or carrier to which anantibody against a cell surface antigen expressed on the desired cellsurface, for instance, an anti-CD8 antibody, is bound, or a method usinga flow cytometer. The above-mentioned carrier is exemplified by magneticbeads or a column. In addition, the cell population rich in the desiredcell can be obtained by removing by adsorbing out cells other than thedesired cell from the culture. For instance, the helper T cell can beremoved from the lymphocyte culture using an antibody against a cellsurface antigen expressed on a surface of the helper T cell, forinstance, an anti-CD4 antibody. In this step, a flow cytometer can bealso used. The cell population rich in the cytotoxic lymphocyte thusobtained has a more potent cytotoxic activity, as compared to that of acell population collected non-selectively from a culture, so that thecell population can be especially preferably used in the medical field.

Further, the present invention provides a cytotoxic lymphocyte obtainedby the method for preparing a cytotoxic lymphocyte of the presentinvention mentioned above. The lymphocyte, especially CTL, has a highcytotoxic activity, which has a characteristic that there is littlelowering of the cytotoxic activity, even when the lymphocyte issubjected to the continuous culture or expansion over a long period oftime. In addition, the present invention provides a medicament(therapeutic agent) comprising the lymphocyte as an effectiveingredient. Especially, the above-mentioned therapeutic agent comprisingthe lymphocyte is suitably used in adoptive immunotherapy. In theadoptive immunotherapy, the lymphocyte having a cytotoxic activitysuitable for treating a patient is administered to the patient by, forinstance, intravenous administration. The therapeutic agent can beprepared by, for instance, blending the lymphocyte prepared by themethod of the present invention as an effective ingredient with, forinstance, a known organic or inorganic carrier suitable for non-oraladministration, an excipient, a stabilizing agent and the like,according to a method known in the pharmaceutical field. Incidentally,various conditions for the therapeutic agent, such as the content oflymphocyte of the present invention in the therapeutic agent and thedose of the therapeutic agent, can be appropriately determined accordingto the known adoptive immunotherapy.

The method for preparing a cytotoxic lymphocyte of the present inventionmay further comprise the step of transducing a foreign gene into thelymphocyte. In other words, one embodiment of the present inventionprovides a method for preparing a cytotoxic lymphocyte, furthercomprising the step of transducing a foreign gene into a cytotoxiclymphocyte. Here, the term “foreign” refers to those which are foreignto a lymphocyte into which a gene is to be transduced.

By carrying out the method for preparing a cytotoxic lymphocyte of thepresent invention, especially the method for expanding a cytotoxiclymphocyte, the DNA replication ability of the cultured lymphocyte isenhanced. Therefore, by including the step of transducing a gene in themethod for preparing a cytotoxic lymphocyte of the present invention,increase in the gene-transducing method is expected.

Methods of transducing a foreign gene are not particularly limited, andan appropriate method can be selected from a known method fortransducing a gene. The step of transducing a gene can be carried out atany given point during the preparation of a cytotoxic lymphocyte. Forinstance, it is preferable to carry out the step simultaneously with anystep of the above-mentioned induction, maintenance and/or expansion ofthe lymphocyte or after the step, from the viewpoint of workingefficiency.

As the above-mentioned method for transducing a gene, any of methodsusing a viral vector, and methods without using the vector can beemployed in the present invention. The details of those methods havebeen already published in numerous literatures.

The above-mentioned viral vector is not particularly limited, and aknown viral vector ordinarily used in the method for transducing a gene,for instance, retroviral vector, lentiviral vector, adenoviral vector,adeno-associated viral vector, simian viral vector, vaccinia viralvector, Sendai viral vector, or the like is used. Especially preferably,as the viral vector, retrovirus, adenovirus, adeno-associated virus orsimian virus is used. As the above-mentioned viral vector, those lackingreplication ability so that the viral vector cannot self-replicate in aninfected cell are preferable.

The retroviral vector is used for the purpose of gene therapy or thelike because there can be stably incorporated a foreign gene insertedinto the vector in chromosomal DNA in the cell into which the vector isto be transduced. Since the vector has a high infection efficiency tothe cell during mitosis and proliferation, the gene transduction ispreferably carried out in the method for preparing a cytotoxiclymphocyte, for instance, the step of expansion.

As the method for transducing a gene without using a viral vector, therecan be employed, but not particularly limited to, for instance, a methodusing a carrier such as liposome or ligand-polylysine, calcium phosphatemethod, electroporation method, particle gun method or the like. In thiscase, there is transduced a foreign gene incorporated into plasmid DNAor linear DNA.

The foreign gene to be transduced into a cytotoxic lymphocyte in thepresent invention is not particularly limited, and an arbitrary genewhich is desired to be transduced into the above-mentioned cell can beselected. As the gene as described above, besides a gene encoding aprotein (for instance, an enzyme, a cytokine, a receptor or the like),for instance, a gene encoding an antisense nucleic acid or a ribozymecan be used. In addition, an appropriate marker gene which is capable ofselecting a cell into which a gene is transduced may be transducedsimultaneously.

The above-mentioned foreign gene can be, for instance, inserted into avector, a plasmid or the like, so that the foreign gene is expressedunder the control of an appropriate promoter, and used. In addition, inorder to achieve an efficient transcription of a gene, there may existin a vector other regulating element which cooperates with a promoter ora transcription initiation site, for instance, an enhancer sequence or aterminator sequence. In addition, for the purpose of inserting a foreigngene into a chromosome of a lymphocyte in which the gene is transducedby homologous recombination, for instance, a foreign gene may bearranged between flanking sequences comprising nucleotide sequences eachhaving homology to nucleotide sequences located on both sides of thedesired target insertion site of the gene in the chromosome. The foreigngene to be transduced may be one that is a naturally occurring or anartificially generated, or may be one in which DNA molecules havingdifferent origins from each other are bound by a known means such asligation. Moreover, the foreign gene may be one having a sequence inwhich a mutation is introduced into a naturally occurring sequencedepending upon its purpose.

According to the method of the present invention, for instance, a geneencoding an enzyme associated with the resistance to a drug used for thetreatment of a patient with cancer or the like is transduced into acytotoxic lymphocyte, thereby giving the lymphocyte a drug resistance.If the cytotoxic lymphocyte as described above is used, adoptiveimmunotherapy and drug therapy can be combined, and, therefore, highertherapeutic effects can be obtained. The drug resistance gene isexemplified by, for instance, a multidrug resistance gene.

On the other hand, conversely to the above-mentioned embodiment, a geneso as to give a sensitivity against a particular drug is transduced intoa cytotoxic lymphocyte, thereby giving sensitivity against the drug. Inthis case, the lymphocyte after being transplanted to a living body canbe removed by administering the drug. The gene for giving sensitivityagainst a drug is exemplified by, for instance, a thymidine kinase gene.

EXAMPLES

The present invention will be more concretely described by means of theexamples, without by no means limiting the scope of the presentinvention thereto.

Preparation Example 1 Preparation of Fibronectin Fragment (1)Preparation of Fibronectin Fragment

H-271, a fragment derived from human fibronectin, was prepared fromEscherichia coli HB101/pHD101 (FERM BP-2264) in accordance with themethod described in U.S. Pat. No. 5,198,423.

In addition, H-296, CH-271 and CH-296, fragments derived from humanfibronectin, were each prepared from a culture obtained by culturingEscherichia coli HB101/pHD102 (FERM BP-7420), Escherichia coliHB101/pCH101 (FERM BP-2799) or Escherichia coli HB101/pCH102 (FERMBP-2800), in accordance with the method described in the above-mentionedgazette.

C-274, a fragment derived from human fibronectin, was prepared from aculture obtained by culturing Escherichia coli JM109/pTF7221

(FERM BP-1915) in accordance with the method described in U.S. Pat. No.5,102,988.

C-CS 1, a fragment derived from human fibronectin, was prepared from aculture obtained by culturing Escherichia coli HB101/pCS25 (FERMBP-5723) in accordance with the method described in Japanese PatentGazette No. 3104178.

CHV-89 and CHV-179, fragments derived from human fibronectin, were eachprepared from a culture obtained by culturing Escherichia coliHB101/pCHV89 (FERM P-12182) or Escherichia coli HB101/pCHV179 (FERMP-12183), in accordance with the method described in Japanese PatentGazette No. 2729712.

In addition, CHV-90, a fragment derived from human fibronectin, wasprepared in accordance with the method described in Japanese PatentGazette No. 2729712. Concretely, a plasmid pCHV90 was constructed inaccordance with the procedures described in the gazette, and thereaftera transformant carrying the plasmid was cultured, and CHV-90 wasprepared from the culture.

CHV-181, a fragment derived from human fibronectin, was prepared byconstructing the plasmid (pCHV181) comprising a DNA encoding CHV-181 inaccordance with the method described in WO 97/18318, thereafterculturing Escherichia coli HB101/pCHV181 into which the plasmid had beenintroduced, and preparing the fragment from the culture in the samemanner as that for the above CHV-179.

(2) Preparation of CHV-92

As to pCHV181, a plasmid for expressing the above-mentioned polypeptideCHV-181, there was constructed a plasmid CHV92 having deletion of aregion encoding a III-13 region in the region encoding CHV-181. Thedeletion procedures were performed in accordance with procedures fordeleting a III-14 coding region from a plasmid pCHV 179, which aredescribed in Japanese Patent Gazette No. 2729712.

Escherichia coli HB101 (Escherichia coli HB101/pCHV92) transformed withthe above-mentioned plasmid pCHV92 was cultured, and the purificationprocedures were carried out in accordance with the method of purifyingthe CHV-89 polypeptide described in Japanese Patent Gazette No. 2729712,to obtain a purified CHV-92 preparation from the resulting culture.

(3) Preparation of H-275-Cys

A plasmid for expressing a polypeptide H-275-Cys was constructed inaccordance with the following procedures. Concretely, a plasmid pCH102was prepared from Escherichia coli HB101/pCH102 (FERM BP-2800). PCR wascarried out using a primer 12S having the nucleotide sequence shown inSEQ ID NO: 20 of Sequence Listing and a primer 14A having the nucleotidesequence shown in SEQ ID NO: 21 of Sequence Listing with the aboveplasmid as a template, to give a DNA fragment of about 0.8 kb, encodinga heparin binding polypeptide of fibronectin. The resulting DNA fragmentwas digested with NcoI and BamHI (both manufactured by Takara Bio Inc.),and thereafter ligated with pTV118N (manufactured by Takara Bio Inc.)digested with NcoI and BamHI, to construct a plasmid pRH1.

A plasmid vector pINIII-ompA₁ [Ghrayeb J. et al., EMBO J., 3(10),2437-2442 (1984)] was digested with BamHI and HincII (manufactured byTakara Bio Inc.) to collect a DNA fragment of about 0.9 kb, containing alipoprotein terminator region. This fragment was mixed and ligated withthe above-mentioned plasmid pRH1 which had been digested with BamHI andHincII, to give a plasmid pRH1-T containing a lac promoter, a DNAfragment encoding a heparin binding polypeptide and a lipoproteinterminator in this order.

The reaction for PCR was carried out by using a primer Cys-A having thenucleotide sequence shown in SEQ ID NO: 22 of Sequence Listing and aprimer Cys-S having the nucleotide sequence shown in SEQ ID NO: 23 ofSequence Listing with this plasmid pRH1-T as a template. Thereafter, thecollected amplified DNA fragment was digested with NotI (manufactured byTakara Bio Inc.), and the DNA fragment was further self-ligated. Acyclic DNA thus obtained was digested with SpeI and ScaI (manufacturedby Takara Bio Inc.) to give a DNA fragment of 2.3 kb, and the resultingfragment was mixed and ligated with a DNA fragment of 2.5 kb, obtainedby digesting the plasmid

pRH1-T with SpeI and ScaI (manufactured by Takara Bio Inc.), to give aplasmid pRH-Cys. The plasmid encodes a polypeptide H-275-Cys in whichfour amino acids Met-Ala-Ala-Ser (residues 1-4 of SEQ ID NO: 19) wereadded to an N-terminal side of the above-mentioned H-271, and furtherCys was added to a C-terminal of the H-271.

The polypeptide H-275-Cys was prepared by the following method.Escherichia coli HB101 transformed with the above-mentioned plasmidpRH-Cys (Escherichia coli HB101/pRH-Cys) was cultured overnight at 37°C. in 120 ml of an LB medium. The cells collected from the culturemedium were suspended in 40 ml of a buffer for disruption (50 mMTris-HCl, 1 mM EDTA, 150 mM NaCl, 1 mM DTT, 1 mM PMSF, pH 7.5), and thesuspension was subjected to ultrasonic treatment to disrupt the cells.The supernatant obtained by centrifugation was subjected toHiTrap-heparin column (manufactured by Pharmacia) which had beenequilibrated with a purifying buffer (50 mM Tris-HCl, pH 7.5). Thenon-adsorbed fraction in the column was washed with the same buffer, andthereafter the elution was carried out with a purifying buffer having a0 to 1 M NaCl concentration gradient. The eluate was analyzed bySDS-polyacrylamide gel electrophoresis, and fractions corresponding to amolecular weight of H-275-Cys were collected to give a purifiedH-275-Cys preparation.

Example 1 Ratio of CD8-Positive Cells in CTLs (1) Isolation and Storageof PBMCs

Blood component was collected from a human normal individual donorhaving HLA-A2.1, obtained with informed consent. The collected bloodcomponent was diluted 2-folds with PBS(−), overlaid on Ficoll-paque(manufactured by Pharmacia), and centrifuged at 500×g for 20 minutes.The peripheral blood mononuclear cells (PBMCs) in the intermediate layerwere collected with a pipette, and washed. The collected PBMCs weresuspended in a storage solution of 90% FBS (manufactured by BioWhittaker)/10% DMSO (manufactured by SIGMA), and stored in liquidnitrogen. During CTL induction, these stored PBMCs were rapidly meltedin water bath at 37° C., and washed with RPMI 1640 medium (manufacturedby Bio Whittaker) containing 10 μg/ml Dnase (manufactured byCalbiochem). Thereafter, the number of living cells was calculated bytrypan blue staining method, and the cells were subjected to eachexperiment.

(2) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was performed bypartially modifying the method of Bednarek et al. [J. Immunology,147(12), 4047-4053 (1991)]. Concretely, PBMCs prepared in item (1) ofExample 2 were suspended in RPMI 1640 medium (manufactured by BioWhittaker) containing 5% human AB-type serum, 0.1 mM nonessential aminoacids, 1 mM sodium pyruvate, 2 mM L-glutamine (hereinabove being allmanufactured by Bio Whittaker), 10 mM HEPES (manufactured by nakalaitesque), 1% streptomycin-penicillin (manufactured by Gibco BRL)(hereinafter simply referred to as “5HRPMI”) so as to have aconcentration of 1 to 4×10⁶ cells/ml. Thereafter, the suspension was puton a 24-well cell culture plate (manufactured by Falcon) in a volume of1 ml/well, and the cells were incubated in a 5% CO₂ wet-type incubatorat 37° C. for 1.5 hours, to separate plastic-adherent monocytes.Thereafter, nonadherent cells were collected using RPMI 1640 medium, andstored on ice as responder cells. To separated monocytes was added 0.5ml each of 5HRPMI containing as an antigen peptide 5 μg/ml epitopepeptide derived from influenza virus protein (A2.1-binding peptidederived from the matrix protein of SEQ ID NO: 24 of Sequence Listing)and 1 μg/ml β2 microglobulin (manufactured by Scrips). The mixture wasincubated at room temperature for 2 hours, and thereafter the cells weresubjected to X-ray irradiation (5500R) to give antigen-presenting cells.The peptide solution was removed by suction from each of the wells, andthe wells were washed with RPMI 1640 medium. Thereafter, the respondercells previously stored on ice were aspirated in 5HRPMI so as to have aconcentration of 0.5 to 2×10⁶ cells/ml, and the suspension was added toantigen-presenting cells in an amount of 1 ml per well. At this time,each fibronectin fragment (hereinafter referred to as “FNfr”) describedin Preparation Example 1 was added so as to have a final concentrationof 10 μg/ml. A group without addition of FNfr was set as the control.The plate was cultured at 37° C. in the presence of 5% CO₂. On thesecond day from the initiation of the culture, 1 ml of 5HRPMI containing60 U/ml IL-2 (manufactured by Shionogi & Co., Ltd.) and 10 μg/ml FNfrwas added to each well (the control containing only IL-2). Also, on thefifth day, a half of the culture supernatant was removed, and 1 ml eachof IL-2- and FNfr-containing medium (the control containing only IL-2),the same as those mentioned above, was added thereto. On the seventhday, the antigen-presenting cells were prepared in the same manner asabove, and thereafter the responder cells which had been cultured forone week were suspended in 5HRPMI so as to have a concentration of 0.5to 2×10⁶ cells/ml. The suspension was added to the antigen-presentingcells prepared in an amount of 1 ml/well each to re-stimulate the cells.At this time, FNfr was added so as to have a final concentration of 10μg/ml (the control being without addition). On the second day fromre-stimulation, 1 ml of 5HRPMI containing 60 U/ml IL-2 and 10 μg/ml FNfrwas added to each well (the control containing only IL-2). Also, on thefifth day, a half of the culture supernatant was removed, and 1 ml eachof the medium having the same content as that before removal was addedthereto. The culture was continued for additional two days, therebyinducing CTLs.

(3) Determination for Cytotoxic Activity of CTLs

The cytotoxic activity of CTLs prepared in item (2) of Example 1 on thefourteenth day after the initiation of induction was evaluated by adetermination method for cytotoxic activity using Calcein-AM [R.Lichtenfels et al., J. Immunological Methods, 172(2), 227-239 (1994)].HLA-A2.1-having EBV transformed B-cells (name of cells: 221A2.1), whichwere cultured overnight together with an epitope peptide or in theabsence of the epitope peptide, were suspended in RPMI 1640 mediumcontaining 5% FBS (manufactured by Bio Whittaker) so as to have aconcentration of 1×10⁶ cells/ml. Thereafter, Calcein-AM (manufactured byDotite) was added to the suspension so as to have a final concentrationof 25 μM, and the cells were cultured at 37° C. for 1 hour. The cellswere washed with a medium not containing Calcein-AM, and thereaftermixed with K562 cells (ATCC CCL-243) in an amount 20 times that of thecells, to give Calcein-labeled target cells. The K562 cells were usedfor excluding nonspecific cytotoxic activity by NK cells admixed in theresponder cells.

The memory CTLs prepared in item (2) of Example 1 were stepwise dilutedwith 5HRPMI so as to have a concentration of from 1×10⁵ to 9×10⁶cells/ml as effector cells. Thereafter, each of the dilutions was pouredinto each well of 96-well cell culture plate in an amount of 100 μl/welleach. Thereto were added the Calcein-labeled target cells prepared tohave a concentration of 1×10⁵ cells/ml in an amount of 100 μl/well each.The plate containing the above-cell suspension was centrifuged at 400×gfor 1 minute, and thereafter incubated in a wet-type CO₂ incubator at37° C. for 4 hours. After 4 hours, 100 μl of the culture supernatant wascollected from each well, and the amount of calcein released(fluorescence intensity) into the culture supernatant was determined byusing fluorescence plate reader (485 nm/538 nm). The cytotoxic activityof the CTLs was calculated by the following equation 1:

Cytotoxic Activity (%)=[(Found Value in Each Well−Minimum ReleasedAmount)/(Maximum Released Amount−Minimum Released Amount)]×100  Equation1:

In the above equation, the minimum released amount is the amount ofcalcein released in the well containing only target cells and K562cells, showing the amount of calcein naturally released from the targetcells. In addition, the maximum released amount refers to the amount ofcalcein released when the cells are completely disrupted by adding 0.1%of the surfactant Triton X-100 (manufactured by nakalai tesque) to thetarget cells. As a result, the cytotoxic activity was inducedimmediately after the induction, but there were hardly any differencesin the cytotoxic activity by the presence or absence of the addition ofFNfr during the induction.

(4) Determination of Content Ratio of CD8-Positive Cells in CellPopulation of CTLs

CTLs which were prepared in item (2) of Example 1 in an amount of 2×10⁵cells were fixed with PBS (manufactured by Nissui) containing 1%paraformaldehyde (manufactured by nakalai tesque), and then washed withPBS. The fixed cells were suspended in 100 μl of PBS containing 1% BSA(manufactured by SIGMA), FITC-labeled mouse IgG1 or FITC-labeled mouseanti-human CD8 antibody (both manufactured by DAKO) was added thereto,and thereafter the mixture was incubated on ice for 30 minutes. Afterthe incubation, the cells were washed with PBS, and suspended again inPBS containing 1% paraformaldehyde. The cells were subjected to flowcytometry using FACS Vantage (manufactured by Becton Dickinson), and thecontent ratio of the CD8-positive cells was determined. The results areshown in Table 1.

TABLE 1 Content Ratio of CD8-Positive Cells Fibronectin Fragment (%)Control (Without Addition of FNfr) 60.2 CH-296 88.8 CH-271 65.7 H-27181.4 C-274 86.2 H-275-Cys 79.0 CHV-89 70.2 CHV-90 77.0 CHV-181 73.1Control (Without Addition of FNfr) 33.0 H-296 40.1 C-CS1 41.6 CHV-9244.0 CHV-179 37.8

As shown in Table 1, in the group with addition of various fibronectinfragments during the CTL induction, the ratio of the CD8-positive cellson the fourteenth day after the initiation of the CTL induction is high,as compared to that of the control without addition of these fibronectinfragments. In other words, it was clarified that the CTLs could beinduced with significantly proliferating the CD8-positive cells by thecopresence of the fibronectin fragment.

Example 2 Induction of Expression of Interleukin-2 Receptor (1)Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of FNfr during the induction.

(2) Determination of Ratio of Interleukin-2 Receptor Expression in CTLs

The ratio of interleukin-2 receptor (IL-2R) expression in the CTLs whichwere prepared in item (1) of Example 2 on the fourteenth day from theinitiation of induction was determined according to the method describedin item (4) of Example 1. Here, in this procedure, an FITC-labeled mouseanti-human CD8 antibody was changed to an FITC-labeled mouse anti-humanIL-2R(CD25) antibody (manufactured by DAKO). The results are shown inTable 2.

TABLE 2 Content Ratio of IL-2R Expression-Positive Cells FibronectinFragment (%) Control (Without Addition of FNfr) 29.8 CH-296 65.9 H-29659.4 H-271 54.6 C-274 61.5 H-275-Cys 78.2 CHV-89 82.3 CHV-90 48.3 CHV-9255.6 CHV-179 50.3 CHV-181 44.8 Control (Without Addition of FNfr) 46.9CH-271 60.9 C-CS1 72.3

As shown in Table 2, in all of CTLs induced with addition of variousfibronectin fragments, an increase in the ratio of IL-2R expression inthe cell population was observed. In other words, it was clarified thatCTLs could be induced with increasing the expression level of IL-2R bycarrying out induction in the copresence of the fibronectin fragment.

Example 3 Expansion of CTLs (1) Induction of Anti-Influenza Virus MemoryCTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of FNfr during the induction.

(2) Expansion of CTLs

CTLs prepared in item (1) of Example 3 were washed with 5HRPMI, and thenmade into a suspension having a concentration of 3×10⁴ cells/ml. On theother hand, allogenic PBMCs not having HLA-A2.1 which were collected inthe same manner as in item (1) of Example 1 were subjected to X-rayirradiation (3300R), and the cells were washed with the medium and thenmade into a suspension having a concentration of 2 to 5×10⁶ cells/mi.These CTLs (3×10⁴ cells) and allogenic PBMCs (4 to 10×10⁶ cells) weresuspended in 10 ml of 5HRPMI, or RPMI 1640 medium (manufactured by BioWhittaker) containing 10% Hyclone FBS, 0.1 mM nonessential amino acids,1 mM sodium pyruvate, 2 mM L-glutamine (all manufactured by BioWhittaker), 10 mM HEPES (manufactured by nakalai tesque) and 1%streptomycin-penicillin (manufactured by Gibco BRL) (hereinafter simplyreferred to as 10HycloneRPMI), and anti-CD3 antibody (manufactured byJanssen-Kyowa) was further added thereto so as to give a finalconcentration of 50 ng/ml. The mixture was placed into a flask of 12.5cm² (manufactured by Falcon), and the cells were cultured in a wet-typeCO₂ incubator at 37° C. for 14 days. During the culture, FNfr was addedso as to have a final concentration of 10 μg/ml which was the same asthat added during the CTL induction. Also, FNfr was not added to acontrol group in which induction was carried out without addition ofFNfr. Stimulation by a peptide was not added at all during this culture.On the first day after the initiation of the expansion, IL-2 was addedso as to have a final concentration of 120 U/ml. Further, on the fourthday and on after the initiation of the culture, procedures of removing ahalf of the culture supernatant, and thereafter adding 5 ml of 5HRPMIcontaining 60 U/ml IL-2 or 10HycloneRPMI to each flask were carried outevery 2 to 3 days. During the culture, FNfr in the same concentrationwas added to the medium for the group with addition of FNfr. On thefourteenth day after the initiation of the expansion, the cytotoxicactivity of CTLs was determined in the same manner as in item (3) ofExample 1. The degree in which the cytotoxic activity before theexpansion is maintained was calculated as “cytotoxic activitymaintenance (%).”

The “cytotoxic activity maintenance (%)” was calculated according to thefollowing equation 2:

Cytotoxic Activity Maintenance (%)=[Cytotoxic Activity (%) AfterExpansion/CytotoxicActivity (%) Before Expansion]×100  Equation 2:

The determination results are shown in Table 3. In the table, an E/Tratio means a ratio of the number of the effector cells (E) to thenumber of the target cells (T).

TABLE 3 Cytotoxic Activity Maintenance (%) Medium Fibronectin FragmentE/T Ratio = 3 5HRPMI Control (Without Addition of FNfr) 17.3 CH-271 53.5H-296 49.3 C-CS1 49.3 CHV-92 66.2 10HycloneRPMI Control (WithoutAddition of FNfr) 48.1 CH-271 250.8 H-296 162.3 H-271 72.2 C-CS1 100.2CHV-92 157.8 Cytotoxic Activity Maintenance (%) Medium FibronectinFragment E/T Ratio = 10 10HycloneRPMI Control (Without Addition of FNfr)46.3 CHV-89 69.0 CHV-90 75.6 Cytotoxic Activity Maintenance (%) MediumFibronectin Fragment E/T Ratio = 3 10HycloneRPMI Control (WithoutAddition of FNfr) 70.4 CH-296 113.5 10HycloneRPMI Control (WithoutAddition of FNfr) 79.3 CHV-179 190.0 CHV-181 94.5

As shown in Table 3, the CTLs of the group with addition of variousfibronectin fragments during the induction and the expansion maintaineda specific, high cytotoxic activity even after the expansion for 14 daysas compared to that of the control without addition of the fibronectinfragment. In other words, it was clarified that the CTLs could beexpanded in a state in which a high cytotoxic activity was maintainedfor a long period of time by carrying out the induction and theexpansion in the copresence of the fibronectin fragment.

Example 4 Expression of IL-2R in Cell Population After Expansion of CTLs(1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of FNfr during the induction.

(2) Determination of Ratio of Interleukin-2 Receptor Expression inExpanded CTLs

The CTLs prepared in item (1) of Example 4 were expanded in the samemanner as in item (2) of Example 3. The ratio of IL-2Rexpression-positive cells was determined for the CTLs after theexpansion thus obtained in the same manner as in item (2) of Example 2.The results are shown in Table 4.

TABLE 4 Content Ratio of IL-2R Expression- Positive Cells FibronectinFragment (%) Control (Without Addition of FNfr) 19.5 CH-271 45.3 H-29647.7 H-271 48.3 C-274 53.5 C-CS 39.7 CHV-891 28.6 CHV-90 60.0 CHV-17953.7 CHV-181 50.3 Control (Without Addition of FNfr) 26.8 CH-296 36.1Control (Without Addition of FNfr) 18.4 H-275-Cys 56.5 CHV-92 59.9

As shown in Table 4, in all of the groups with addition of variousfibronectin fragments during the induction and the expansion of CTLs, anincrease in the ratio of IL-2R expressing cells in the cell populationwas observed.

In other words, it was clarified that CTLs could be expanded withincreasing the expression level of IL-2R by carrying out induction andexpansion of CTLs in the presence of the fibronectin fragment.

Example 5 Induction and Expansion of CTLs in the Presence of Fibronectin(1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was performed inaccordance with the method described in item (2) of Example 1 using thePBMCs isolated and stored in accordance with the method described initem (1) of Example 1. During the induction, fibronectin (manufacturedby Calbiochem) was added in place of FNfr so as to have a finalconcentration of 10 μg/ml (a control being without addition). Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofthe induction was determined in the same manner as in item (3) ofExample 1. As a result, there were hardly any differences in thecytotoxic activity by the presence or absence of the addition offibronectin during the induction.

(2) Determination of Ratio of Interleukin-2 Receptor Expression in CTLs

The ratio of IL-2R expression-positive cells was determined for the CTLsprepared in item (1) of Example 5 in the same manner as in item (2) ofExample 2. The results are shown in Table 5.

TABLE 5 Content Ratio of IL-2R Expression- Positive Cells Fibronectin(%) Control (Without Addition of Fibronectin) 34.0 Fibronectin 64.6

As shown in Table 5, in CTLs induced in the presence of fibronectin, anincrease in the expression level of IL-2R in the cell population wasobserved.

In other words, it was clarified that CTLs could be induced withincreasing the expression level of IL-2R by carrying out induction ofCTLs in the presence of the fibronectin.

(3) Expansion of CTLs

CTLs prepared in item (1) of Example 5 were expanded in the same manneras in item (2) of Example 3. During the expansion, to the group withaddition of fibronectin during the induction, fibronectin (manufacturedby Calbiochem) was added, so as to have a final concentration of 10μg/ml (a control without addition). The cytotoxic activity of the CTLsobtained was determined in the same manner as that of item (3) ofExample 1, and the degree in which the cytotoxic activity before theexpansion is maintained was calculated as “cytotoxic activitymaintenance (%).”

The determination results are shown in Table 6.

TABLE 6 Cytotoxic Activity Maintenance (%) Fibronectin E/T Ratio = 3Control (Without Addition of Fibronectin) 48.1 Fibronectin 148.9

As shown in Table 6, the group in which the induction and the expansionof CTLs were carried out in the presence of fibronectin maintained ahigh cytotoxic activity. On the other hand, the cytotoxic activity ofthe control without addition of fibronectin during the induction and theexpansion of CTLs was clearly lowered. In other words, it was clarifiedthat CTLs could be expanded in a state in which a specific cytotoxicactivity was maintained for a long period of time by adding fibronectinduring the induction and the expansion of CTLs.

Example 6 Expansion of CTLs in the Presence of Immobilized Fibronectin(FN) Fragment (1) Immobilization of FN Fragment

A fibronectin fragment was immobilized to a culture equipment (vessel)used in the following experiment. Concretely, PBS containing variousfibronectin fragments (final concentration: 10 μg/ml) was added in anamount of 1 to 2 ml each to a 24-well cell culture plate and a 12.5 cm²flask. The plate and the flask were subjected to incubation at roomtemperature for 5 hours, and then stored at 4° C. until use. Inaddition, the plate and the flask were washed twice with PBS before use.

(2) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs Ls was performed inaccordance with the method described in item (2) of Example 1 using thePBMCs isolated and stored in accordance with the method described initem (1) of Example 1. During the induction, a plate immobilized withFNfr was used as a culture equipment (for a control, a plate withoutimmobilization treatment was used). The cytotoxic activity of CTLs afterthe induction was evaluated in the same manner as in item (3) ofExample 1. As a result, there were hardly any differences in thecytotoxic activity by the presence or absence of immobilization of FNfrto the plate used during the induction.

(3) Expansion of CTLs

The CTLs prepared in item (2) of Example 6 were expanded in the samemanner as in item (2) of Example 3. During the expansion, flasks withvarious FNfr's immobilized thereto were used as culture equipments (fora control, a flask without immobilization treatment was used). Inaddition, 10HycloneRPMI was used as a medium.

The degree in which the cytotoxic activity of CTLs thus expanded wasmaintained as compared to that before the expansion was evaluated as“cytotoxic activity maintenance (%).”

The determination results are shown in Table 7.

TABLE 7 Cytotoxic Activity Maintenance (%) Fibronectin Fragment E/TRatio = 3 Control (Without Immobilization of FNfr) 48.1 CH-271 95.4H-296 95.0 H-271 133.9 C-CS1 73.8 H-275-Cys 137.7 CHV-92 92.7 Control(Without Immobilization of FNfr) 18.7 CH296 67.4 C-CS1 78.5 CHV-89 90.8CHV-90 73.0 CHV-179 112.5 CHV-181 25.6

As shown in Table 7, in the group in which the culture equipment (plate,flask) immobilized with the fibronectin fragment was used during theinduction and the expansion of CTLs, the CTLs maintained a specific,high cytotoxic activity even after the expansion. On the other hand, inthe control in which the equipment without immobilization with thefibronectin fragment was used during the induction and the expansion ofCTLs, the cytotoxic activity was clearly lowered. In other words, it wasclarified that the CTLs could be expanded in a state in which a highcytotoxic activity was maintained for a long period of time, comparableto that of the fragment dissolved in the medium, by using theimmobilized fibronectin fragment.

Example 7 Content Ratio of CD8-Positive Cells in Cell Population AfterExpansion of CTLs (1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of FNfr during the induction.

(2) Determination of Content Ratio of CD8-Positive Cells in ExpandedCTLs

The CTLs prepared in item (1) of Example 7 were expanded in the samemanner as in item (2) of Example 3. The content ratio of CD8-positivecells was determined for the CTLs after the expansion thus obtained inthe same manner as in item (4) of Example 1. The results are shown inTable 8.

TABLE 8 Content Ratio of CD8-Positive Cells Fibronectin Fragment (%)Control (Without Addition of FNfr) 40.9 CH-296 85.1 CH-271 72.1 H-27183.9 Control (Without Addition of FNfr) 75.4 H-296 87.2 C-CS1 86.5Control (Without Addition of FNfr) 33.4 CHV-90 72.9 CHV-92 51.6 CHV-17957 CHV-181 63.5

As shown in Table 8, in all of the groups with addition of variousfibronectin fragments during the induction and the expansion of CTLs, anincrease in the content ratio of CD8-positive cells in the cellpopulation after the expansion was observed.

In other words, it was clarified that CTLs could be expanded withsignificantly proliferating CD8-positive cells by carrying out inductionand expansion of CTLs in the presence of the fibronectin fragment.

Example 8 Induction of Interleukin-2 Receptor Expression in CTLs Inducedin the Presence of Immobilized Fibronectin Fragment (1) Immobilizationof FN Fragment

A fibronectin fragment was immobilized to a culture equipment (vessel)in the same manner as in item (1) of Example 6.

(2) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was performed inaccordance with the method described in item (2) of Example 1 using thePBMCs isolated and stored in accordance with the method described initem (1) of Example 1. During the induction, a plate immobilized withFNfr prepared in item (1) of Example 8 was used as a culture equipment(for a control, a plate without immobilization treatment was used). Thecytotoxic activity of CTLs after the induction was evaluated in the samemanner as in item (3) of Example 1. As a result, there were hardly anydifferences in the cytotoxic activity by the presence or absence ofimmobilization of FNfr to the plate used during the induction.

(3) Expansion of CTLs

The CTLs prepared in item (2) of Example 8 were expanded in the samemanner as in item (2) of Example 3. During the expansion, a flask withFNfr immobilized thereto prepared in item (1) of Example 8 was used asculture equipments (for a control, a flask without immobilizationtreatment was used). In addition, 10HycloneRPMI was used as a medium.

The ratio of IL-2R expression-positive cells was determined in the samemanner as in item (2) of Example 2 for the CTLs before and after theexpansion thus obtained.

The determination results are shown in Table 9.

TABLE 9 Content Ratio of IL- Content Ratio of 2R Expression- IL-2RExpression- Positive Cells Positive Cells Before Expansion AfterExpansion Fibronectin Fragment (%) (%) Control (Without 14.4 6.8Immobilization of FNfr) CH-296 68.1 34.0 CH-271 28.3 14.7 H-296 21.322.9 C-274 30.3 20.5 C-CS1 56.8 34.1 H-275-Cys 43.6 17.2 CHV-89 34.636.8 CHV-90 47.3 29.1 CHV-92 37.2 13.0 CHV-179 52.3 16.3 CHV-181 37.418.3

As shown in Table 9 for CTLs, in the group in which the cultureequipment (plate, flask) immobilized with the fibronectin fragment wasused during the induction and the expansion of CTLs, an increase in aratio of IL-2R expression was observed as compared to that of thecontrol group in both before and after the expansion. In other words, itwas clarified that the CTLs could be expanded with maintaining a highIL-2R expression level, comparable to that of the fragment dissolved inthe medium, by using the immobilized fibronectin fragment.

Example 9 Induction of Interleukin-2 Receptor Expression on Surface ofCD8 Cells (1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of FNfr during the induction.

(2) Determination of Ratio of Interleukin-2 Receptor Expression in CTLs

The ratio of interleukin-2 receptor (IL-2R) expression in the CTLs(especially on a surface of CD8 cells) prepared in item (1) of Example 9on the fourteenth day from initiation of induction was determinedaccording to the method described in item (4) of Example 1. Here, inthis procedure, an FITC-labeled mouse anti-human CD8 antibody was usedas a primary antibody, and a PE-labeled mouse anti-human IL-2R(CD25)antibody (manufactured by DAKO) was used as a secondary antibody. Theresults are shown in Table 10.

TABLE 10 Content Ratio of CD8/IL-2R- Double Positive Cell PopulationFibronectin Fragment (%) Control (Without Addition of FNfr) 30.7 CH-29656.8

As shown in Table 10, in the CTLs induced with addition of variousfibronectin fragments, an increase in the ratio of IL-2R expression inthe CD8-positive cell population was observed. In other words, it wasclarified that CTLs could be induced with increasing the expressionlevel of IL-2R on the surface of CD8 cells by carrying out induction inthe copresence of the fibronectin fragment.

Example 10 Content Ratios of CD8-Positive Cells in Cell PopulationBefore and After Expansion of CTLs

(Comparison of Fibronectin with Fibronectin Fragment)

(1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of fibronectin and FNfrduring the induction.

(2) Determination of Content Ratio of CD8-Positive Cells in ExpandedCTLs

The CTLs prepared in item (1) of Example 10 were expanded in the samemanner as in item (2) of Example 3. The content ratio of CD8-positivecells was determined for the CTLs before and after the expansion thusobtained in the same manner as in item (4) of Example 1. The results areshown in Table 11.

TABLE 11 Content Ratio of Content Ratio of CD8-Positive CellsCD8-Positive Cells Before Expansion After Expansion (%) (%) Control 50.531.2 (Without Addition of FNfr) Fibronectin 67.3 22.5 H-271 75.1 51.3CHV-90 71.3 43.5

As shown in Table 11, in all of the groups with addition of variousfibronectin fragments during the induction and the expansion of CTLs, anincrease in the content ratio of CD8-positive cells in the cellpopulation before the expansion and after the expansion was observed ascompared to that of the control group.

In other words, it was clarified that CTLs could be favorably expandedwith significantly proliferating CD8-positive cells in both before theexpansion and after the expansion by carrying out induction andexpansion of CTLs in the presence of the fibronectin fragment ascompared to that of fibronectin per se.

Example 11 Induction of IL-2R Expression in Cell Population Before andAfter Expansion of CTLs

(Comparison of Fibronectin with Fibronectin Fragment)

(1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of fibronectin and FNfrduring the induction.

(2) Determination of Content Ratio of II-2R Expression-Positive Cells inExpanded CTLs

The CTLs prepared in item (1) of Example 11 were expanded in the samemanner as in item (2) of Example 3. The content ratio of IL-2Rexpression-positive cells was determined for the CTLs before and afterthe expansion thus obtained in the same manner as in item (2) of Example2. The results are shown in Table 12.

TABLE 12 Content Ratio of Content Ratio of IL-2R Expression- IL-2RExpression- Positive Cells Before Positive Cells Expansion AfterExpansion (%) (%) Control 34.0 15.3 (Without Addition of FNfr)Fibronectin 64.6 50.6 H-271 76.6 76.8

As shown in Table 12, in all of the groups with addition of fibronectinfragments during the induction and the expansion of CTLs, an increase inthe content ratio of IL-2R expression-positive cells in the cellpopulation before the expansion and after the expansion was observed ascompared to that of the control group. This increase ratio wassignificantly high as compared to the group with the addition offibronectin.

In other words, it was clarified that CTLs could be favorably expandedwith significantly proliferating IL-2R expression-positive cells in bothbefore the expansion and after the expansion by carrying out inductionand expansion of CTLs in the presence of the fibronectin fragment ascompared to that of fibronectin per se.

Example 12 Expansion of CTLs (Comparison of Fibronectin with FibronectinFragment) (1) Induction of Anti-Influenza Virus Memory CTLs

The induction of anti-influenza virus memory CTLs was carried out in thesame manner as in item (2) of Example 1 using the PBMCs which wereisolated and stored in the same manner as in item (1) of Example 1. Thecytotoxic activity of CTLs on the fourteenth day after the initiation ofinduction was evaluated in the same manner as in item (3) of Example 1.As a result, there were hardly any differences in the cytotoxic activityby the presence or absence of the addition of fibronectin and FNfrduring the induction.

(2) Expansion of CTLs

The CTLs prepared in item (2) of Example 12 were expanded in the samemanner as in item (2) of Example 3. In addition, 10HycloneRPMI was usedas a medium.

The degree in which the cytotoxic activity of CTLs thus expanded wasmaintained as compared to that before the expansion was evaluated as“cytotoxic activity maintenance (%).”

The determination results are shown in Table 13.

TABLE 13 Cytotoxic Activity Maintenance (%) E/T Ratio = 3 Control(Without Addition of FNfr)  48.1 Fibronectin 148.9 CH-271 250.8

As shown in Table 13, the CTLs of the group with addition of fibronectinfragment during the induction and the expansion maintained a specific,high cytotoxic activity even after the expansion for 14 days as comparedto that of the control group without addition of fibronectin fragment.Also, its activity was significantly high as compared to the group withthe addition of fibronectin.

In other words, it was clarified that the CTLs could be favorablyexpanded in a state in which a high cytotoxic activity was maintainedfor a long period of time by carrying out the induction and theexpansion in the copresence of the fibronectin fragment as compared tothat in the copresence of the fibronectin per se.

Example 13 Determination of Expansion Fold in Culture System of LAKCells (Lymphokine-Activated Killer Cells) (1) Immobilization ofAnti-Human CD3 Antibody and FN or FN Fragment

An anti-human CD3 antibody and fibronectin or FN fragment wereimmobilized to a culture equipment (vessel) used in the followingexperiment. Concretely, 1 ml (in a case of a 24-well plate) or 2 ml (ina case of 12.5 cm² flask) each of PBS containing an anti-human CD3antibody (manufactured by Janssen-Kyowa) (final concentration 5 μg/ml)was added to a 24-well cell culture plate or a 12.5 cm² cell cultureflask (manufactured by Falcon). During the addition, fibronectin or eachof the fibronectin fragments (FNfr) listed in Preparation Example 1 wasadded to the group with addition of fibronectin or FN fragment so as tohave a final concentration of 10 μg/ml (in the case of the

24-well plate) or 25 μg/ml (in the case of the 12.5 cm² flask). As acontrol, there was also set a group without addition of fibronectin andFNfr.

After these culture equipments were incubated at room temperature for 5hours, the culture equipments were stored at 4° C. until use.Immediately before use, PBS containing the antibody and FNfr was removedby aspiration from these culture equipments, and thereafter each wellwas washed twice with PBS, and then once with XVIVO20 medium(manufactured by Bio Whittaker) containing 5% human AB type serum(manufactured by Bio Whittaker) and 1% streptomycin-penicillin(manufactured by GIBCO BRL) (hereinafter simply referred to as5HXVIVO20), and the culture equipments were subjected to eachexperiment.

(2) Induction and Culture of LAK Cells

PBMCs which were prepared in item (1) of Example 1 were suspended in5HXVIVO20 so as to have a concentration of 0.5 to 1×10⁶ cells/ml, andthereafter the suspension was put on a plate immobilized with theanti-human CD3 antibody, or a plate immobilized with the anti-human CD3antibody and fibronectin or FNfr prepared in item (1) of Example 13 in avolume of 1 ml/well, and IL-2 (manufactured by Shionogi & Co., Ltd.) wasadded thereto so as to have a final concentration of 1000 U/ml. Theseplates were subjected to culture at 37° C. in 5% CO₂ (zeroth day ofculture). On the second and third days from the initiation of culture,5HXVIVO20 containing 1000 U/ml IL-2 was added in a volume of 1 ml/well.On the fourth day from the initiation of culture, a culture mediumproperly diluted with 5HXVIVO20 was transferred to a fresh flask towhich nothing was immobilized, and IL-2 was added so as to have a finalconcentration of 500 U/ml. The culture was continued, the culture mediumwas properly diluted with 5HXVIVO20 every 2 or 3 days in the same manneras the fifth day from the initiation of culture, and IL-2 was added soas to have a final concentration of 300 to 500 U/ml. On the seventh tofifteenth day from the initiation of culture, the number of living cellswas counted by trypan blue staining method, and calculated as anexpansion fold by comparing the number with the number of cells at theinitiation of culture. The results are shown in Table 14.

TABLE 14 Expansion Number of Fold Cultured Days FN/FN Fragment (folds) 7 Days Control (Without Immobilization of FN/FNfr) ×103  Fibronectin×233  CH-296 ×218  H-296 ×247   9 Days Control (Without Immobilizationof FN/FNfr) ×250  Fibronectin ×1190 CH-296 ×1286 H-296 ×1075 11 DaysControl (Without Immobilization of FN/FNfr) ×576  Fibronectin ×2304CH-296 ×1728 H-296 ×2088 Control (Without Immobilization of FN/FNfr)×660  C-CS1 ×1170 15 Days Control (Without Immobilization of FN/FNfr)×1980 Fibronectin ×3348 CH-296 ×5364 Control (Without Immobilization ofFN/FNfr) ×2906 C-CS1 ×5117

As shown in Table 14, in the group using the culture equipmentimmobilized with each of the fibronectin fragments at an early stage ofthe induction of LAK cells, the expansion fold of LAK cells is high ascompared to that of the control group. In addition, the expansion foldof the group immobilized with each of the fibronectin fragments washigher than the group using the culture equipment immobilized withfibronectin on the fifteenth day from the initiation of culture.Therefore, it was clarified that in a case where the expansion iscarried out over a long period of time, induction and culture of LAKcells are suitably carried out in an even higher expansion fold by thecopresence of the fibronectin fragment at an early stage of theinduction of LAK cells, as compared to that of fibronectin per se.

Example 14 Determination of Proliferation Ratio in Culture System of LAKCells (1) Induction and Culture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as in item (2) of Example 13. The proliferation ratio of thecells from the fourth day to the seventh day from the initiation ofculture during this stage was calculated. The results are shown in Table15.

TABLE 15 Number of Proliferation Cells at Ratio from Initiation 4th Dayto of Culture Fibronectin Fragment 7th Day (folds) 5 × 10⁵ cells/mlControl (Without Immobilization of FNfr)  2.7 folds CH-296 16.9 foldsControl (Without Immobilization of FNfr) 33.5 folds H-296 49.5 folds 1 ×10⁶ cells/ml Control (Without Immobilization of FNfr)  6.2 folds CH-29620.4 folds Control (Without Immobilization of FNfr) 23.5 folds H-29643.5 folds

As shown in Table 15, in the group using the culture equipmentimmobilized with each of the fibronectin fragments at an early stage ofthe induction of LAK cells, the proliferation ratio of LAK cells fromthe fourth day to the seventh day of the initiation of culture is highas compared to that of the control group. In other words, it wasclarified that LAK cells could be induced and cultured at a fasterproliferation rate by the copresence of the fibronectin fragment at anearly stage of the induction of LAK cells.

Example 15 Determination of Expansion Fold in Culture System of LAKCells (Induction and Culture of LAK Cells from Low Number of Cells) (1)Induction and Culture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (2) of Example 13. During this stage, the cellconcentration at the initiation of culture was adjusted so as to have aconcentration of 2×10⁵ to 1×10⁶ cells/ml (1×10⁵ to 5×10⁵ cells/cm²). Onthe fifteenth day from the initiation of culture, the number of livingcells was counted by trypan blue staining method, and an expansion foldwas calculated as compared to that with the number of cells at theinitiation of culture. The results are shown in Table 16.

TABLE 16 Number of Cells Expansion at Initiation Fold of CultureFibronectin Fragment (folds) 2 × 10⁵ cells/ml Control (WithoutImmobilization of FNfr) ×48.6   (1 × 10⁵ cells/cm²) CH-296 ×1004  5 ×10⁵ cells/ml Control (Without Immobilization of FNfr) ×438   (2.5 × 10⁵cells/cm²)  CH-296 ×1094  1 × 10⁶ cells/ml Control (WithoutImmobilization of FNfr) ×1020   (5 × 10⁵ cells/cm²) CH-296 ×1476 

As shown in Table 16, in the group using the culture equipmentimmobilized with each of the fibronectin fragments during the inductionof LAK cells, a high expansion fold was obtained on the fifteenth dayfrom the initiation of culture, regardless of the number of cells at theinitiation of culture. By contrast, in the control group, the expansionfold on the fifteenth day from the initiation of culture was low whenthe number of cells at the initiation of culture was low. In otherwords, it was clarified that LAK cells could be induced and cultured ata high expansion fold by the copresence of the fibronectin fragmentduring the induction of LAK cells from the low number of cells,regardless of the number of cells at the initiation of culture.

Example 16 Determination of Expansion Fold in Culture System of LAKCells (Induction and Culture of LAK Cells from Low Number ofCells/Culture Without Procedures of Dilution) (1) Induction and Cultureof LAK Cells

PBMCs which were prepared in item (1) of Example 1 were suspended in5HXVIVO20 so as to have a concentration of 1×10⁴ cells/ml, andthereafter the suspension was put on a plate immobilized with theanti-human CD3 antibody, or a E-well plate immobilized with theanti-human CD3 antibody and fibronectin or FNfr prepared in the samemanner as in item (1) of Example 13 in a volume of 1 ml/well. Fourmilliliters of 5HXVIVO20 was added thereto (1×10³ cells/cm²), and IL-2(manufactured by Shionogi & Co., Ltd.) was added thereto so as to have afinal concentration of 500 U/ml. These plates were subjected to cultureat 37° C. in 5% CO₂ (zeroth day of culture). On the second, third andfourth days from the initiation of culture, IL-2 was added so as to havea final concentration of 500 U/ml. The culture was continued, and IL-2was added every two to three days so as to have a final concentration of500 U/ml from the seventh day and on from the initiation of culture.During the addition, the dilution procedure of the culture medium wasnot carried out at all.

On the fifteenth day from the initiation of culture, the number ofliving cells was counted by trypan blue staining method, and calculatedas an expansion fold by comparing the number with the number of cells atthe initiation of culture. The results are shown in Table 17.

TABLE 17 Number of Expansion Cultured Fold Days FN/FN Fragment (folds)15 Days Control (Without Immobilization of FN/FNfr) ×15 Fibronectin ×628CH-296 ×773 H-296 ×960

As shown in Table 17, in the group using the culture equipmentimmobilized with each of the fibronectin fragments during the inductionof LAK cells from the low number of cells, a high expansion fold wasobtained on the fifteenth day from the initiation of culture, withoutrequiring the dilution procedure of the cells during the course of theinduction. Also, this expansion fold was high even when compared to thegroup using the culture equipment immobilized with the fibronectin. Bycontrast, in the control group, the cells hardly proliferated even onthe fifteenth day from the initiation of culture. In other words, it wasclarified that LAK cells could be induced and cultured at a highexpansion fold by the copresence of the fibronectin or fibronectinfragment, preferably the fibronectin fragment, during the induction ofLAK cells from the low number of cells, without requiring the dilutionprocedure at all.

Example 17 Induction of IL-2R Expression in LAK Cells (1) Induction andCulture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (2) of Example 13.

(2) Determination of Ratio of IL-2R Expression in LAK Cells

The ratio of IL-2R expression in the LAK cells which were subjected toinduction and culture in item (1) of Example 17 was determined accordingto the method described in item (2) of Example 2. The results are shownin Table 18. In the table, the content ratio of IL-2Rexpression-positive cells (%) is shown as ratio of IL-2R expression (%).

TABLE 18 Ratio of IL-2R Number of Expression Cultured Days FN/FNFragment (%) 4 Days Control (Without Immobilization of FN/FNfr) 86.5Fibronectin 97.2 CH-296 97.6 H-296 97.7 C-CS1 94.9 7 Days Control(Without Immobilization of FN/FNfr) 59.3 Fibronectin 77.6 CH-296 90.4H-296 89.1 C-CS1 65.8

As shown in Table 18, in the group using the culture equipmentimmobilized with each of the fibronectin fragments at an early stage ofthe induction of LAK cells, a high ratio of IL-2R expression wasobtained on the surface of LAK cells during the culture. Also, thisratio of IL-2R expression was high even when compared to the group usingthe culture equipment immobilized with the fibronectin. In other words,it was clarified that LAK cells could be induced and cultured with aratio of IL-2R expression being favorably higher than that of thefibronectin per se by the copresence of the fibronectin fragment duringthe induction of LAK cells.

Example 18 Induction of IL-2R Expression in LAK Cells (1) Induction andCulture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (2) of Example 13.

(2) Determination of Ratio of IL-2R Expression in LAK Cells

The ratio of IL-2R expression on the surfaces of CD4 cells and CD8 cellsin the LAK cells which were induced and cultured in item (1) of Example18 on the seventh day was determined according to the method describedin item (2) of Example 9. Here, in this procedure, an FITC-labeled mouseanti-human CD4 antibody or an FITC-labeled mouse anti-human CD8 antibodywas used as a primary antibody, and a PE-labeled mouse anti-humanIL-2R(CD25) antibody was used as a secondary antibody. The results areshown in Table 19.

TABLE 19 Content Ratio of Content Ratio of CD4/IL-2R CD8/IL-2R DoublePositive Double Positive Cells Cells Fibronectin Fragment (%) (%)Control (Without Immobilization 20.5 49.4 of FN/FNfr) CH-296 41.2 61.6C-CS1 24.4 54.6

As shown in Table 19, in the group using the culture equipmentimmobilized with each of the fibronectin fragments at an early stage ofthe induction of LAK cells, a high ratio of IL-2R expression could beinduced on the surface of LAK cells (both CD4-positive and CD8-positivecells) during the culture. In other words, it was clarified that LAKcells could be induced and cultured with a high ratio of IL-2Rexpression on the cell surfaces of both CD4-positive and CD8-positivecells by the copresence of the fibronectin fragment during the inductionof LAK cells.

Example 19 Content Ratio of CD8-Positive Cells in LAK Cell Population(1) Induction and Culture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (2) of Example 13.

(2) Determination of Content Ratio of CD8-Positive Cell Population inLAK Cells

The content ratio of CD8-positive cells in the LAK cells which wereinduced and cultured in item (1) of Example 19 on the fifteenth day wasdetermined according to the method described in item (4) of Example 1.The results are shown in Table 20.

TABLE 20 Content Ratio of CD8-Positive Cells Fibronectin Fragment (%)Control (Without Immobilization of FN/FNfr) 42.9 CH-296 72.1 H-296 76.0

As shown in Table 20, in the group using the culture equipmentimmobilized with each of the fibronectin fragments at an early stage ofinduction of LAK cells, a high content ratio of CD8-positive cells couldbe induced in LAK cells during the culture. In other words, it wasclarified that LAK cells could be induced and cultured with a highcontent ratio of CD8-positive cells in LAK cells by the copresence ofthe fibronectin fragment during the induction of LAK cells.

Example 21 Determination of Expansion Fold in Culture System of LAKCells (1) Induction and Culture of LAK Cells

PBMCs which were prepared in item (1) of Example 1 were suspended in5HXVIVO20 so as to have a concentration of 0.5 to 1×10⁶ cells/ml, andthereafter the suspension was put on a plate immobilized with theanti-human CD3 antibody, or a plate immobilized with the anti-human CD3antibody and FNfr prepared in item (1) of Example 13 in a volume of 1ml/well, and IL-2 (manufactured by Shionogi & Co., Ltd.) was addedthereto so as to have a final concentration of 1000 U/ml. These plateswere subjected to culture at 37° C. in 5% CO₂ (zeroth day of culture).On the second and third days from the initiation of culture, 5HXVIVO20containing 1000 U/ml IL-2 was added in a volume of 1 ml/well. On thefourth day from the initiation of culture, a culture medium properlydiluted with 5HXVIVO20 was transferred to a fresh flask to which nothingwas immobilized, and IL-2 was added so as to have a final concentrationof 500 U/ml. On the eighth or ninth day from the initiation of culture,a culture medium properly diluted with 5HXVIVO20 was transferred to aflask immobilized with the anti-human CD3 antibody, or a flaskimmobilized with the anti-human CD3 antibody and FNfr (provided that theconcentration of the anti-human CD3 antibody used in the immobilizationwas 0.5 μg/ml) prepared in the same manner as in item (1) of Example 13,and IL-2 was added so as to have a final concentration of 500 U/ml. Onthe eleventh day or twelfth day from the initiation of culture, aculture medium properly diluted again with 5HXVIVO20 was transferred toa fresh flask to which nothing was immobilized, and IL-2 was added so asto have a final concentration of 500 U/ml. On the fifteenth day from theinitiation of culture, the number of living cells was counted by trypanblue staining method, and calculated as an expansion fold by comparingthe number with the number of cells at the initiation of culture. Theresults are shown in Tables 21 and 22. In the table, “Donor” denotesPBMC donors.

TABLE 21 Stimulation on Stimulation on 0th Day from 8th Day fromExpansion Fibronectin Initiation of Initiation of Fold Donor FragmentCulture Culture (folds) A Control Anti-CD3 None ×80 (Without Anti-CD3Anti-CD3 ×38 Immo- bilization of FNfr) CH-296 Anti-CD3 + CH-296 None×1452 Anti-CD3 + CH-296 Anti-CD3 ×1620 Anti-CD3 + CH-296 Anti-CD3 +CH-296 ×2700 B Control Anti-CD3 None ×710 (Without Anti-CD3 Anti-CD3×2363 Immo- bilization of FNfr) CH-296 Anti-CD3 + CH-296 None ×504Anti-CD3 + CH-296 Anti-CD3 ×5468 Anti-CD3 + CH-296 Anti-CD3 + CH-296×14243 C Control Anti-CD3 None ×1805 (Without Anti-CD3 Anti-CD3 ×4200Immo- bilization of FNfr) CH-296 Anti-CD3 + CH-296 Anti-CD3 + CH-296×35700 H-296 Anti-CD3 + H-296 Anti-CD3 + H-296 ×16950

TABLE 22 Stimulation on Stimulation on 0th Day from 9th Day fromExpansion Fibronectin Initiation of Initiation of Fold Donor FragmentCulture Culture (folds) B Control (Without Anti-CD3 None ×2074Immobilization of FNfr) Anti-CD3 Anti-CD3 ×2880 CH-296 Anti-CD3 + CH-296Anti-CD3 + CH-296 ×38400 CH-271 Anti-CD3 + CH-271 Anti-CD3 + CH-271×12672 H-296 Anti-CD3 + H-296 Anti-CD3 + H-296 ×67584 H-271 Anti-CD3 +H-271 Anti-CD3 + H-271 ×8755 C-274 Anti-CD3 + C-274 Anti-CD3 + C-274×8525 C-CS1 Anti-CD3 + C-CS1 Anti-CD3 + C-CS1 ×9677 CHV-90 Anti-CD3 +CHV-90 Anti-CD3 + CHV-90 ×10138 CHV-179 Anti-CD3 + CHV-179 Anti-CD3 +CHV-179 ×8294 CHV-181 Anti-CD3 + CHV-181 Anti-CD3 + CHV-181 ×5760

As shown in Tables 21 and 22, in the group using the culture equipmentin which each of the fibronectin fragments and the anti-CD3 antibodywere repeatedly immobilized at an early stage and an intermediate stageof the induction of LAK cells, an expansion fold of LAK cells is high ascompared to that of the control group. These expansion ratios were farhigher than the expansion fold in the group using the culture equipmentin which only the anti-CD3 antibody was repeatedly immobilized at anearly stage and an intermediate stage of the induction of LAK cells. Inother words, it was clarified that LAK cells could be induced andcultured with a higher expansion fold by stimulation using thefibronectin fragment and the anti-CD3 antibody at an early stage and anintermediate stage of induction of LAK cells.

Example 21 Determination of Proliferation Ratio in Culture System of LAKCells (1) Induction and Culture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (1) of Example 20. The proliferation ratio of thecells from the fourth day to the eighth day from the initiation ofculture and the proliferation ratio of the cells from the eleventh dayto the fifteenth day from the initiation of culture during theprocedures were calculated. The results are shown in Tables 23 and 24.

TABLE 23 Proliferation Stimulation on Stimulation on Ratio from 0th Dayfrom 8th Day from 11th to 15th Fibronectin Initiation of Initiation ofDays Donor Fragment Culture Culture (folds) A Control Anti-CD3 None  6.7folds (Without Anti-CD3 Anti-CD3  8.3 folds Immo- bilization of FNfr)CH-296 Anti-CD3 + CH-296 None  2.6 folds Anti-CD3 + CH-296 Anti-CD3  5.5folds Anti-CD3 + CH-296 Anti-CD3 + CH-296 11.1 folds B Control Anti-CD3None  7.4 folds (Without Anti-CD3 Anti-CD3 17.5 folds Immo- bilizationof FNfr) CH-296 Anti-CD3 + CH-296 None  0.9 folds Anti-CD3 + CH-296Anti-CD3 19.8 folds Anti-CD3 + CH-296 Anti-CD3 + CH-296 60.3 folds CControl Anti-CD3 None  5.2 folds (Without Anti-CD3 Anti-CD3 22.2 foldsImmo- bilization of FNfr) CH-296 Anti-CD3 + CH-296 Anti-CD3 + CH-29694.0 folds H-296 Anti-CD3 + H-296 Anti-CD3 + H-296 35.0 folds

TABLE 24 Proliferation Stimulation on Stimulation on Ratio from 0th Dayfrom 9th Day from 11th to 15th Fibronectin Initiation of Initiation ofDays Donor Fragment Culture Culture (folds) B Control Anti-CD3 None  5.7folds (Without Anti-CD3 Anti-CD3 15.6 folds Immo- bilization of FNfr)CH-296 Anti-CD3 + Anti-CD3 + 55.6 folds CH-296 CH-296 CH-271 Anti-CD3 +Anti-CD3 + 25.0 folds CH-271 CH-271 H-296 Anti-CD3 + H-296 Anti-CD3 +H-296 88.9 folds H-271 Anti-CD3 + H-271 Anti-CD3 + H-271 23.8 foldsC-274 Anti-CD3 + C-274 Anti-CD3 + C-274 61.7 folds C-CS1 Anti-CD3 +C-CS1 Anti-CD3 + C-CS1 28.0 folds CHV-90 Anti-CD3 + Anti-CD3 + 44.0folds CHV-90 CHV-90 CHV-179 Anti-CD3 + Anti-CD3 + 32.7 folds CHV-179CHV-179 CHV-181 Anti-CD3 + Anti-CD3 + 41.7 folds CHV-181 CHV-181

As shown in Tables 23 and 24, in the group using the culture equipmentin which each of the fibronectin fragments and the anti-CD3 antibodywere repeatedly immobilized at an early stage and an intermediate stageof induction of LAK cells, the proliferation ratio of LAK cells was highin a later stage of the induction as compared to that of the control.These proliferation ratios were far higher than the proliferation ratioof LAK cells in the later stage of the induction in the group using theculture equipment in which only the anti-CD3 antibody was repeatedlyimmobilized at an early stage and an intermediate stage of induction ofLAK cells. In other words, it was clarified that LAK cells could beinduced and cultured with a higher proliferation ratio by stimulationusing the fibronectin fragment and the anti-CD3 antibody at an earlystage and an intermediate stage of induction of LAK cells.

Example 22 Induction of IL-2R Expression in LAK Cells (1) Induction andCulture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (1) of Example 20.

(2) Determination of Ratio of IL-2R Expression in LAK Cells

The ratio of IL-2R expression in the LAK cells which were subjected toinduction and culture in item (1) of Example 22 on the fifteenth day wasdetermined according to the method described in item (2) of Example 2.The results are shown in Tables 25 and 26. In the tables, the contentratio of the IL-2R expression-positive cells (%) is shown as ratio ofIL-2R expression (%).

TABLE 25 Stimulation on Stimulation on Ratio of 0th Day from 9th Dayfrom IL-2R Fibronectin Initiation of Initiation of Expression FragmentCulture Culture (%) Control (Without Anti-CD3 None  4.8 ImmobilizationAnti-CD3 Anti-CD3 32.6 of FNfr) CH-296 Anti-CD3 + CH-296 None  2.5Anti-CD3 + CH-296 Anti-CD3 72.3 Anti-CD3 + CH-296 Anti-CD3 + CH-296 94.7H-296 Anti-CD3 + H-296 None  1.4 Anti-CD3 + H-296 Anti-CD3 50.2Anti-CD3 + H-296 Anti-CD3 + H-296 89.6

TABLE 26 Stimulation on Stimulation on Ratio of 0th Day from 9th Dayfrom IL-2R Fibronectin Initiation of Initiation of Expression FragmentCulture Culture (%) Control (Without Anti-CD3 None  4.8 ImmobilizationAnti-CD3 Anti-CD3 18.0 of FNfr) CH-296 Anti-CD3 + Anti-CD3 + 84.0 CH-296CH-296 CH-271 Anti-CD3 + Anti-CD3 + 67.1 CH-271 CH-271 H-296 Anti-CD3 +H-296 Anti-CD3 + H-296 79.9 H-271 Anti-CD3 + H-271 Anti-CD3 + H-271 51.6C-274 Anti-CD3 + C-274 Anti-CD3 + C-274 66.4 C-CS1 Anti-CD3 + C-CS1Anti-CD3 + C-CS1 72.5 CHV-90 Anti-CD3 + Anti-CD3 + 52.6 CHV-90 CHV-90CHV-179 Anti-CD3 + Anti-CD3 + 63.4 CHV-179 CHV-179 CHV-181 Anti-CD3 +Anti-CD3 + 68.3 CHV-181 CHV-181

As shown in Tables 25 and 26, in the group using the culture equipmentin which each of the fibronectin fragments and the anti-CD3 antibodywere repeatedly immobilized at an early stage and an intermediate stageof the induction of LAK cells, the ratio of IL-2 expression on thesurface of LAK cells on the fifteenth day after the initiation ofculture was high as compared to that of the control. These ratios ofIL-2R expression were far higher than the ratios of IL-2R expression inthe group using the culture equipment in which only the anti-CD3antibody was repeatedly immobilized at an early stage and anintermediate stage of the induction of LAK cells. In other words, it wasclarified that LAK cells could be induced and cultured with a higherratio of IL-2R expression by stimulation using the fibronectin fragmentat an early stage and an intermediate stage of the induction of LAKcells.

Example 23 Determination of Ratio of CD8-Positive Cells in LAK Cells (1)Induction and Culture of LAK Cells

The induction and the culture of LAK cells were carried out in the samemanner as that of item (1) of Example 20.

(2) Determination of Content Ratio of CD8-Positive Cells in LAK CellPopulation

The content ratio of CD8-positive cells in the LAK cell population whichwere subjected to induction and culture in item (1) of Example 23 on thefifteenth day was determined according to the method described in item(4) of Example 1. The results are shown in Table 27.

TABLE 27 Stimulation on Stimulation on Content Ratio 0th Day from 8thDay from of CD8-Positive Fibronectin Initiation of Initiation of CellsFragment Culture Culture (%) Control (Without Anti-CD3 None 42.9Immobilization Anti-CD3 Anti-CD3 55.2 of FNfr) CH-296 Anti-CD3 + CH296None 72.1 Anti-CD3 + CH296 Anti-CD3 85.2 Anti-CD3 + CH296 Anti-CD3 +75.9 CH-296 H-296 Anti-CD3 + H296 None 76.0 Anti-CD3 + H296 Anti-CD382.0 Anti-CD3 + H296 Anti-CD3 + 77.1 H296

As shown in Table 27, in the group using the culture equipment in whicheach of the fibronectin fragments and the anti-CD3 antibody wererepeatedly immobilized at an early stage and an intermediate stage ofthe induction of LAK cells, the content ratio of CD8-positive cells inthe LAK cell population on the fifteenth day from the initiation ofculture was high as compared to that of the control. These contentratios of CD8-positive cells were far higher than the content ratios ofCD8-positive cells in the group using the culture equipment in whichonly the anti-CD3 antibody was repeatedly immobilized at an early stageand an intermediate stage of the induction of LAK cells. In other words,it was clarified that LAK cells could be induced and cultured with ahigher content ratio of CD8-positive cells by stimulation using thefibronectin fragment at an early stage and an intermediate stage of theinduction of LAK cells.

SEQUENCE LISTING FREE TEXT

SEQ ID NO: 1; Partial region of fibronectin named III-8.SEQ ID NO: 2; Partial region of fibronectin named III-9.SEQ ID NO: 3; Partial region of fibronectin named III-10.SEQ ID NO: 4; Partial region of fibronectin named III-12.SEQ ID NO: 5; Partial region of fibronectin named III-13.SEQ ID NO: 6; Partial region of fibronectin named III-14.SEQ ID NO: 7; Partial region of fibronectin named CS-1.SEQ ID NO: 8; Fibronectin fragment named C-274.SEQ ID NO: 9; Fibronectin fragment named H-271.SEQ ID NO: 10; Fibronectin fragment named H-296.SEQ ID NO: 11; Fibronectin fragment named CH-271.SEQ ID NO: 12; Fibronectin fragment named CH-296.SEQ ID NO: 13; Fibronectin fragment named C-CS1.SEQ ID NO: 14; Fibronectin fragment named CHV-89.SEQ ID NO: 15; Fibronectin fragment named CHV-90.SEQ ID NO: 16; Fibronectin fragment named CHV-92.SEQ ID NO: 17; Fibronectin fragment named CHV-179.SEQ ID NO: 18; Fibronectin fragment named CHV-181.SEQ ID NO: 19; Fibronectin fragment named H-275-Cys.

SEQ ID NO: 20; Primer 12S. SEQ ID NO: 21; Primer 14A. SEQ ID NO: 22;Primer Cys-A. SEQ ID NO: 23; Primer Cys-S.

SEQ ID NO: 24; Designed peptide based on matrix protein derived frominfluenza virus.

INDUSTRIAL APPLICABILITY

According to the process for preparing a cytotoxic lymphocyte of thepresent invention, there is obtained a cytotoxic lymphocyte in which ahigh cytotoxic activity is maintained, an expression level of IL-2R issignificantly increased, and a ratio of a CD8-positive cell is improved.The lymphocyte is suitably used, for instance, in adoptiveimmunotherapy. Therefore, there is expected a great contribution of theprocess of the present invention to the medical field.

1. A method for expanding cytotoxic lymphocytes which comprises:culturing precursor cells, capable of differentiating to cytotoxiclymphocytes, wherein the precursor cells are selected from the groupconsisting of peripheral blood mononuclear cells and blood componentscontaining these cells in the presence of at least one recombinantfibronectin fragment together with interleukin-2 and an anti-CD3antibody, wherein the recombinant fibronectin fragment is a polypeptidecomprising an amino acid sequence selected from the group consisting ofSEQ ID NOS: 8 to 19, wherein the expanded cytotoxic lymphocytes maintaincytotoxic activity longer than cytotoxic lymphocytes expanded in theabsence of at least one fibronectin fragment, and wherein the expandedcytotoxic lymphocytes express more CD8 than cytotoxic lymphocytesexpanded in the absence of at least one fibronectin fragment.
 2. Themethod according to claim 1, wherein the expanded cytotoxic lymphocyteshighly express an interleukin-2 receptor at a higher level thancytotoxic lymphocytes expanded in the absence of at least onefibronectin fragment.
 3. The method according to claim 1, wherein saidat least one recombinant fibronectin fragment is immobilized on a solidphase.
 4. The method according to claim 3, wherein the solid phase is acell culture vessel or a cell culture carrier.
 5. The method accordingto claim 1, further comprising transducing a foreign gene into thecytotoxic lymphocytes.
 6. The method according to claim 5, wherein theforeign gene is transduced using a retrovirus, adenovirus,adeno-associated virus or simian virus.
 7. The method according to claim1, wherein an expansion ratio of the cytotoxic lympohocytes is high ascompared to that of a method for expanding cytotoxic lymphocytes in theabsence of at least one recombinant fibronectin fragment.
 8. The methodaccording to claim 1, wherein the precursor cells are cultured with therecombinant fibronectin fragment for four days after initiation ofculture.
 9. A method for increasing the number of CD8 positive cells ina population of cytotoxic lymphocytes, which comprises: culturingprecursor cells, capable of differentiating to cytotoxic lymphocytes,wherein the precursor cells are selected from the group consisting ofperipheral blood mononuclear cells and blood components containing thesecells in the presence of at least one recombinant fibronectin fragmenttogether with interleukin-2 and an anti-CD3 antibody, thereby increasingthe number of CD8-positive cells in the cultured cells, wherein therecombinant fibronectin fragment is a polypeptide comprising an aminoacid sequence selected from the group consisting of SEQ ID NOS: 8 to 19,wherein the cultured cytotoxic lymphocytes maintain cytotoxic activitylonger than cytotoxic lymphocytes expanded in the absence of at leastone recombinant fibronectin fragment.
 10. The method according to claim9, wherein the precursor cells are cultured with the recombinantfibronectin fragment for four days after initiation of culture.